Vet Pathol 2014 Ramos Vara 42-87-4

Article

When Tissue Antigens and AntibodiesGet Along: Revisiting the Technical Aspectsof Immunohistochemistry—The Red,Brown, and Blue Technique

J. A. Ramos-Vara1 and M. A. Miller1

AbstractOnce focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of abroad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to anindividual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immuno-histochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixationtechniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer,standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and otherhigh-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results.It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissuecollection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification andinterlaboratory comparison of IHC results.

Keywordsantibodies, antigen retrieval, fixation, immunohistochemistry, review, standardization, technical aspects, validation

Paul Ehrlich introduced the term antibody (Antikorper) in189146 and hypothesized that cell surface receptors bind specif-ically to toxins in a lock-and-key interaction. However, it wasnot until 1941 that antigen detection in tissue sections wasreported, marking the birth of immunohistochemistry (IHC).65

Since then, the number of tests and their specificity andsensitivity have increased to the point that IHC routinely sup-plements the morphologic approach to pathology.292 Althougha major early application was in the characterization ofneoplasms, IHC currently has broader and more clinicallyoriented applications in diagnosis, prognosis, therapeutic deci-sion making, and pathogenesis.206,372 In essence, IHC fills thegap between classic histopathology and molecular pathology.

Immunohistochemistry bridges 3 disciplines: immunology,histology, and chemistry. The fundamental concept is thedemonstration of antigens within tissue sections by means ofspecific antibodies. The immunoglobulin molecule has bindingsites for antigens and for other antibodies (Fig. 1). Antigen-antibody binding is demonstrated with a colored histochemicalreaction visible by light or fluorescent microscopy.284 In otherwords, the basic principle of IHC, as with other ‘‘special’’histochemical methods, is visual localization of cell or tissuetarget molecules based on a satisfactory signal-to-noise ratio.371

Although conceptually simple, the methodology of IHC hasbecome more complex with more stringent requirements for

sensitivity and specificity.237 The initial simple (direct) methodsproduced quick results but lacked sensitivity. Currently,extremely sensitive methods can detect 1 or multiple antigenssimultaneously or can screen hundreds of tissues in the samesection (tissue microarray technology) for the presence of aparticular antigen. The feasibility of detecting multiple antigensby IHC has improved dramatically with the use of multispectralanalysis.209 Another critical advance in the 1990s was theintroduction of techniques to ‘‘retrieve’’ antigens that had beenaltered by fixation, increasing exponentially the number of anti-gens detectable in routinely fixed tissues.284

In some cases (eg, prion diseases), IHC is considered the goldstandard to which other techniques are compared. In contrast tomany detection techniques, IHC allows colocalization of an anti-gen with a lesion, thereby dramatically enhancing diagnosticinterpretation and understanding of the pathogenesis.

1 Department of Comparative Pathobiology, College of Veterinary Medicine,Purdue University, West Lafayette, IN, USA

Corresponding Author:J. A. Ramos-Vara, Animal Disease Diagnostic Laboratory and Department ofComparative Pathobiology, Purdue University, 406 South University, WestLafayette, IN 47907, USA.Email: [email protected]

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Figure 1. Immunoglobulin structure. The Fc fragment is delimited by the oval dotted line; the F(ab) fragment, by the oval dashed line. The areawithin the square is the variable region of the F(ab) fragment. Figure 2. Effects of autolysis on immunohistochemistry (IHC). Parathyroidadenoma; dog. Diffuse nonspecific background reactivity in autolyzed tissue, including nuclear labeling (with a cytoplasmic marker) in thyroidfollicular cells (upper inset) and parathyroid chief cells (lower inset). Thyroid gland (asterisk). Parathyroid adenoma (solid circle).Immunoperoxidase-DAB for cytokeratins, hematoxylin counterstain. Figure 3. Effects of overfixation. (a, b) IHC for Bartonella sp in equine fetallung. Strong immunoreactivity at 2 days’ fixation (a) with loss of reactivity at 11 weeks’ fixation (b). (c, d) IHC for cytokeratins; skin, dog. Strongimmunoreactivity at 2 days’ fixation (c); weak reactivity at 7 weeks (d). Immunoperoxidase-DAB, hematoxylin counterstain. Figure 4. Effects ofsuboptimal fixation on IHC. B-cell lymphoma; lymph node, dog. Only the outer rim of tissue was adequately fixed (formalin). Proper hematoxylinand eosin (HE) staining (a) and IHC reactivity are limited to the well-fixed portion of the section. Immunoperoxidase-DAB for CD79a. Figure 5.Effects of alcohol fixation on protein structure. Alcohol interacts with hydrophobic moieties, modifying the tertiary structure of the protein.Modified from Ramos-Vara284 with permission from Veterinary Pathology.

Ramos-Vara and Miller 43



Although IHC has become a routine tool for veterinarydiagnostic and research studies, the variable cross-reactivityof antibodies among different species raises many challengesfor the comparative pathologist.284 This review addressestechnical aspects of IHC—fixation, antigen retrieval,antigen-antibody reactions, detection methods, controls,standardization, validation, and quality assurance—as wellas interpretation. Basic aspects of immunocytochemistry willbe addressed briefly.

Overview of the Immunohistochemical Test

The IHC technique is a combination of immunologic andchemical reactions visualized with a photonic microscope. Thetechnique can be divided into 3 phases (Table 1). Phase 1 (prea-nalytical) starts with sample procurement, followed by tissuefixation, trimming, and embedding, and ending with tissuesectioning on a microtome. Phase 2 (analytical) starts with depar-affination of tissue sections; includes preincubation steps (eg,antigen retrieval, blocking of nonspecific activities), incubationwith the primary antibody, and labeling of the antigen-antibodyreaction; and ends with slide counterstaining and coverslipping.Phase 3 (postanalytical) includes interpretation of results andgeneration of an IHC report, as well as evaluation of the IHCcontrols.369

Preanalytical Phase of IHC

Fixation

Fixation of tissues is necessary to (1) adequately preserve cel-lular components, including soluble and structural proteins; (2)

prevent autolysis and displacement of cell constituents, includ-ing antigens and enzymes; (3) stabilize cellular materialsagainst deleterious effects of subsequent procedures; and(4) facilitate conventional staining and immunostaining.142

No fixative optimally fulfills all these goals.284 Chemical fixa-tion is generally used for diagnostic IHC; the most commonchemical fixative is formalin (Table 2).

Effects of Delayed Fixation. Prompt transfer of postmortem spe-cimens into fixative is critical.149 Delayed fixation candecrease the mitotic index, with up to 40% reduction whenfixation is delayed more than 12 hours;70,82,151 this reductionin mitotic index can alter tumor grade.356 Likewise, withbiopsy specimens, once the tissue is removed from the livingbody, biochemical alterations include adenosine triphosphate(ATP) depletion and disruption of sodium, potassium, andcalcium gradients.149 Prolonged hypoxia as a result ofdelayed fixation can cause cellular swelling, generation ofreactive oxygen species, and activation of various enzymes,all of which can change the immunoreactivity of the targetantigen, especially for measurement of apoptosis or detectionof the phosphorylation state of signaling pathways.94,149

Delayed fixation can affect the IHC results in variousways.79,94,149,223,272 A fixation delay of more than 2 hourscan decrease the detection of breast cancer biomarkers suchas estrogen receptor (ER), progesterone receptor (PR), andHER2,186 although others have not observed differences inER and PR reactivity in samples stored in similar conditions(4!C) for several days.5 Enzyme-rich tissues, such as intes-tine or pancreas, autolyze rapidly; proteolysis can lead toincreased background staining (Fig. 2).149 Diffusion of

Table 1. Steps and Variables in an Immunohistochemical Test.

Steps Variables

Preanalytical phase Sample procurement Delayed fixation, prolonged ischemia, thickness of sampleFixation Cross-linking vs coagulating fixatives, durationDecalcification Type of decalcification solution and durationTissue processing Paraffin-embedded vs frozen tissuesTissue sectioning Thickness of tissue section, drying temperature and duration, tissue section aging

Analytical phase Deparaffination Dewaxing agentAntigen retrieval Detergents, enzymes, HIERBlocking nonspecific

reactivitiesEndogenous enzymes, hydrophobic binding, pigments

Primary antibody Monoclonal vs polyclonal, Ag recognition (native vs linear), specificity, speciesvariability

Detection system Avidin-biotin vs polymer-based systems, ultrasensitive methodsEnzyme-substrate-

chromogenColor detection

Multiplex IHC Enzyme-substrate combinationsCounterstain Contrast between chromogen and counterstain

Postanalytical phase Control performance Animal species compatibility, tissue processingInterpretation Pathologist vs automated evaluationReport Percentage of positive cells, positive vs negative threshold, stand-alone test vs

ancillary testDiagnostic, prognostic, or theranostic test

HIER, heat-induced antigen retrieval; IHC, immunohistocehmistry.

44 Veterinary Pathology 51(1)



soluble proteins with fixation delay has been reported withthyroglobulin, myoglobin, glial fibrillary acidic protein(GFAP), and other cellular proteins.95,184,355 Protozoaand fungi may be more resistant to autolysis than viruses.291

If delay in fixation is anticipated, samples should berefrigerated.149

The quality of fixation is influenced by the type of fixa-tive; fixative pH, buffers, concentration, osmolality, andadditives; fixation time and temperature; and the use ofpostfixation procedures (eg, decalcification). Two types offixatives are used in histopathology: cross-linking (noncoa-gulating) and coagulating. The choice of fixative determinesthe need for pretreatments (eg, antigen retrieval), titer of theprimary antibody, the intensity of the specific reaction ver-sus background (signal-to-noise ratio), and even the detec-tion pattern of antigens.149,264

Formaldehyde: A Cross-Linking Fixative. Formaldehyde is thestandard fixative for routine histology and IHC. Two factors

contribute to the use of formaldehyde as the fixative of choicefor most histologic procedures: (1) for more than a century,pathologists have studied tissues fixed in formalin and havebecome accustomed to histologic examination through theartifacts it produces, and (2) archived paraffin blocks, aninvaluable resource for disease studies, most commonly con-tain formalin-fixed tissues.85 Formaldehyde preserves mainlypeptides and the general structure of cellular organelles. It alsointeracts with nucleic acids but has little effect on carbohy-drates.91,194 It is a good preservative of lipids if the fixativecontains calcium.176

The Chemistry of Formalin Fixation. Fixation with formaldehydeis a 3-step process of penetration, covalent bonding, andcross-linking.51,77 Formation of cross-links between targetpeptides and irrelevant proteins reduces or blocks theirimmunoreactivity.39 Whereas these steps happen simultane-ously, they do so at different rates, with penetration beingabout 12 times faster than bonding and the latter 4 times faster

Table 2. Comparison of Fixatives Used in Immunohistochemistry.

Fixative Description Fixation Times Advantages Disadvantages

Formalin 10% neutral bufferedformalin

>8 hours Most common fixative Fixation (cross-linking) is slow

12–36 hours necessary forcomplete fixation

Quick penetration Ag cross-linking results in need forAg retrieval

Special handling and disposalrequirements

Zincformalin

Mixture of zinc sulfateand formalin

4–48 hours Shorter fixation times thanformalin

Possible quenching of primaryfluorescence

May not need antigenretrieval

Special handling and disposalrequirements

Preserves tissue morphologywell

Alcohol/acetone

70%, 90% EtOH or90% EtOH/10%acetone

Variable; often occurs intissue processingsecondary to formalinfixation

Shorter fixation times Unknown archival stability

10–15 minutes for cryostatsections/cytology smears

Good preservation ofcytoplasmic intermediatefilaments

Shrinking or hardening of the tissue

Bouin’s Mixture of formalinand picric acid

1–12 hours Rapid tissue fixation Reduced preservation of manyantigens, particularly lipid-containing antigens

Useful for endocrine tissuesand tumors

Fixation may result in brittle tissuesdifficult to section

Postfixation procedure to removepicric acid

Needs special handling and disposalrequirements

B-5a Mixture of mercuricchloride, sodiumacetate, and formalin

1–6 hours Used mostly in lymphoidtissue due to enhancedcytological detail andimmunoreactivity ofimmunoglobulins andintracytoplasmic antigens

Tissue hardeningSurface immunoglobulins not well

preservedSpecial handling and disposal

requirements

Modified and used with permission by the Clinical and Laboratory Standards Institute, document I/LA28-A2.149

aFixatives containing mercury require special handling and disposal procedures.




than cross-linking.51 For example, a 3-mm-thick sample willbe 100% penetrated, 24% bonded, and 6% cross-linked in 8hours; after 24 hours of fixation, it will be 70% bonded and36% cross-linked.53 Fixation at 37!C is faster than at25!C.145 Notably, formaldehyde penetrates tissues ratherquickly, but tissue penetration does not equal tissue fixation.Tissue fixation by formaldehyde is considered a clockreaction because formaldehyde is in equilibrium with methyleneglycol.149 Only formaldehyde (and not methylene glycol) pro-duces the characteristic cross-linking formaldehyde fixa-tion.109,149 As formaldehyde is used during tissue fixation,methylene glycol is converted into formaldehyde to maintainthe equilibrium; this newly formed formaldehyde reacts withproteins, producing additional cross-links and therefore furtherfixation.149 In solution, formaldehyde binds the followingamino acids: lysine, tyrosine, asparagine, tryptophan, histidine,arginine, cysteine, and glutamine.235,261,334 Therefore, knowingthe amino acid composition of an epitope may predict its sen-sitivity to fixation in formalin.348 However, the cross-linkingbetween antigens and unrelated tissue proteins also has amarked effect on the immunoreactivity of antigens afterfixation.347 Cross-linking due to aldehyde fixation can producenew epitopes that specifically react with antibodies designedfor another purpose (eg, the specific detection of enamelproteins in glutaraldehyde-fixed tissue by an antibody tovimentin).178

The basic mechanism of fixation with formaldehyde is theformation of addition products (adducts) between the formalinand uncharged reactive amino groups (-NH or NH2) that even-tually will form cross-links.75 The formation of methyloladducts inactivates nucleases and proteases.261 Once theaddition product (reactive hydroxymethyl compound) isformed, additional cross-links develop. Thus, in the presenceof a second reactive hydrogen, the hydroxymethyl group formsa methylene bridge.

1. Formation of addition products

Protein-H + Protein-CH2OH

Reactive hydrogenon tissue

Formaldehyde Reactive hydroxymethylcompound

addition product

CH2O

2. Formation of methylene bridges

2Protein-CH OH + Protein-H

Reactive hydroxymethylcompound

addition product

Second reactive hydrogenon the protein

Protein-CH2-Protein + 2O

Methylene bridge cross-link

H

The final result of formaldehyde fixation is a profound changein the conformation of macromolecules, which may make therecognition of proteins (antigens) by antibodies difficult or

impossible.245 Although the primary and secondary structuresof proteins are relatively spared, formalin fixation alters the 3-dimensional (tertiary and quaternary) structure of pro-teins.142,231 Changes in the tertiary structure may not be thedirect result of formaldehyde fixation but rather the subsequentinteraction of cross-linked proteins with ethanol or clearingagents during tissue processing.77,108,261 More energy (eg,antigen retrieval) is needed to reverse formalin fixationcross-links after processing tissues in ethanol, confirming thatantigen immunoreactivity is affected not only by formalin fixa-tion but also by postfixation procedures.108

The deleterious effects of formaldehyde on immunoreac-tivity in cells and tissues can be partially reversed,92 whereasglutaraldehyde fixation is considered irreversible.91 Pro-longed washing of fixed tissues reduces further fixation byremoving unbound formaldehyde,142 although establishedcross-links remain.92 Long-term storage of formalin-fixedtissues in alcohol halts the formation of cross-links and, there-fore, facilitates antigen detection should the tissues be neededsubsequently for IHC. Overfixation can also be partially cor-rected by soaking tissues in concentrated ammonia plus 20%chloral hydrate.210

The pH of a fixative buffer dramatically influences thedegree of cross-linking.92,113,282 Amino acids are charged(-NH3

þ) at lower pH and uncharged (-NH2) at higher pH.When using neutral buffered formalin, the pH is shifted toneutrality, causing dissociation of hydrogen ions from thecharged amino groups (-NH3

þ) of the protein side chains,resulting in uncharged amino groups (-NH2). Theseuncharged groups contain hydrogen ions that can react withformalin to form addition groups and cross-links. In otherwords, the use of 10% buffered formalin (pH 6.8–7.2) pro-duces more cross-links than nonbuffered formalin and there-fore more deleterious effects for IHC.284 If the fixative isacidic formalin, cross-linking is reduced, so the antigen retrie-val procedure may need modification. In comparison, 10%neutral buffered formalin, 10% formal saline, and 10% zincformalin were each superior to 10% formal acetic in preser-ving antigenicity.412

After an initial drop in immunoreactivity with fixation, thereis a plateau of variable duration before antigens become unrec-ognizable by specific antibodies.347 Overfixation could producefalse-negative results due to excessive cross-links,142 especiallybefore the advent of heat-based antigen retrieval methods.205

However, significant reduction of immunoreactivity is detectedin only a few markers after several weeks’ fixation (Fig.3),238,403,404 so it seems that the negative effects of overfixationare antigen and antibody dependent and can be overcome inmany instances with an appropriate antigen retrieval proce-dure.6,22,164,242 Vimentin was once used as an internal controlof antigenicity loss in overfixed tissues.18,306 However, withheat-based antigen retrieval, some antigens in overfixed tissuesmay not be well preserved even if vimentin reactivity does notindicate antigen degradation.6 The effect of prolongedformaldehyde fixation on an antigen depends on its cellular loca-lization142—for example, there is irreversible loss of Bcl-2




immunoreactivity in the nucleus, even with heat-based antigenretrieval, whereas cytoplasmic immunoreactivity is preservedor increased after antigen retrieval.154

Underfixation can also produce unexpected results and isconsidered a more common and serious problem than overfixa-tion.35,149 In a typical diagnostic setting, formalin-fixed tissuesare processed through a series of alcohol gradients prior toparaffin embedding. If large samples are fixed only 24 to 48hours or small biopsy specimens for only several hours,cross-links may develop only in the periphery of the specimenwith the core of the tissue left unfixed or fixed only by coagu-lation with the alcohol used for dehydration during tissueprocessing,142,405 resulting in a IHC gradient across the section(Fig. 4).149 Underfixation can produce irrelevant or equivocalresults that can alter the detection or scoring of some biomar-kers.123,171 Antigen retrieval of underfixed tissues may resultin unexpected antigen detection.284 Underfixed tissues areeasily damaged by harsh antigen retrieval methods, with subse-quent loss of antigenicity.303

There is no optimal fixation time for every antigen. Thestructure of the antigen as well as its relationship with otherproteins probably influences the effect of fixation on immu-noreactivity. The current Clinical and Laboratory StandardsInstitute recommendation for formalin fixation of diagnosticspecimens is 16 to 32 hours; on average, complete fixationis achieved after 24 to 48 hours.149 Interestingly, the recom-mended 10:1 ratio of fixative to sample was challenged whena 2:1 ratio and 48-hour fixation at room temperature wasconsidered sufficient for routine histology and IHC.53 Fixa-tive penetration, bonding, and cross-linking are faster athigher temperatures. Samples to be fixed should not bethicker than 2 to 4 mm. Although formaldehyde has beendeemed a suboptimal fixative for IHC, with optimal antigenretrieval procedures, it is satisfactory for most antigens.

Glyoxal, the Ideal Formalin Substitute? Glyoxal is a dialdehydethat resembles 2 back-to-back formaldehyde molecules.76

Whereas formaldehyde forms a single hydrated species(methylene glycol), glyoxal has several hydrated forms, themost common of which is 1,3-dioxolane.76 One advantage ofglyoxal over formaldehyde is that it does not emit vapors. Inaddition, glyoxal has poor reactivity toward most end groupsand forms addition compounds only with arginine, lysine,cysteine, and the single a-amine group of proteins, whereasformaldehyde reacts at room temperature (RT) with almost allend groups that contain nitrogen or oxygen to form single-carbon adducts.76 In comparison to formaldehyde fixation,glyoxal fixation is faster with minimal or no cross-linking,rendering antigen retrieval unnecessary for many antigens.Although a special antigen retrieval solution with high pH canbe used, standard antigen retrieval procedures in glyoxal-fixed tissues may be ineffective or even deleterious.76 Theperceived advantages of glyoxal were disputed, however, ina quantitative image analysis study in which formaldehydewas considered superior in terms of morphologic preservationand IHC signal.59

Coagulative Fixatives. The problems with formaldehyde fixation,especially the loss of immunoreactivity (particularly before thedevelopment of heat-based antigen retrieval methods), haveprompted a search for alternative fixatives.284 Many formalinsubstitutes are coagulating fixatives that precipitate proteinsby breaking hydrogen bonds without protein cross-linking. Themost common types of coagulative fixatives are dehydrants(alcohols and acetone) and strong acids (picric acid, trichloroa-cetic acid). Most body fluid proteins have hydrophilic moietiesin contact with water and hydrophobic moieties in closercontact with each other, stabilizing hydrophobic bonding.Removal of water by ethanol destabilizes protein hydrophobicbonding because the hydrophobic areas are released from therepulsion of water, and the protein tertiary structure unfolds(Fig. 5).92 Simultaneously, removal of water destabilizeshydrogen bonding in hydrophilic areas. The resulting proteindenaturation causes inadequate cellular preservation142,153,401

and a possible shift in intracellular immunoreactivity asreported for some growth factor peptides and cytokines(Fig. 6).42,383 Coagulative fixation maintains tissue structureat the light microscopic level fairly well but results in cytoplas-mic flocculation as well as poor preservation of mitochondriaand secretory granules.284

The Search for Alternatives to Formaldehyde Fixation. There is no‘‘one fixative fits all’’ for IHC.128 Formaldehyde is used mainlybecause it is reliable for general histology, it is inexpensive,and its deleterious effects can often be countered with antigenretrieval.280 In addition, the interpretation of retrospectivestudies would be onerous if archived paraffin blocks containedtissues preserved in a variety of fixatives over the years. Thevalue of nonformaldehyde fixatives for research purposes hasbeen demonstrated for various antigens.* Most availableformalin substitutes are alcohol solutions; therefore, fixationis based on dehydration and protein coagulation.51,83,198

Weigners fixative, a mixture of alcohols and pickling salt,appears to be superior to formaldehyde for DNA and RNAstudies, performed comparably to formaldehyde for IHC, andmaintained immunoreactivity for some biomarkers afterprolonged fixation when formaldehyde did not.193 Carnoy’sfixative, a mixture of alcohol–chloroform–acetic acid, wassuperior to formalin for IHC detection of some antigenswithout antigen retrieval in tissue-engineered constructs.195

Tissues fixed in PAXgene, which consists of a fixation reagent(methanol, acetic acid, and a organic solvent) and a postfixa-tion stabilization reagent (alcohol mixture), may not need anti-gen retrieval for some antigens,183 although immunoreactivityfor other antigens is reduced compared with formalin-fixed,paraffin-embedded (FFPE) tissues.21 For molecular analysis,PAXgene preserves RNA better than does formalin.130

The immunoreactivity of antigens in fixed and paraffin-embedded tissue varies markedly depending on the antigen and

*References 75, 91, 103, 118, 171, 193, 198, 241, 244, 262, 280, 317, 322, 352,

432, 434.




more specifically on the epitope recognized by the antibody.8,59

In a comparison of the effect of different fixatives (strongcross-linkers, weak cross-linkers, coagulant, and combinationcoagulant/cross-linkers) on antigen immunoreactivity in mousetissue, formaldehyde, followed by the combination fixatives,performed better than the other types.59 Substitution of a differ-ent fixative in an IHC test validated for formalin-fixed tissuedemands a new validation study.149

Microwave Fixation. The use of a microwave can reduce fixationtime and therefore the overall time for tissue processing. Afterimmersing the tissue in formalin at RT for at least 4 hours,adequate fixation can be achieved in a 5-mm-thick sample bymicrowaving in formalin solution for 1.5 to 4 minutes at55!C.149 Validation of microwave fixation procedures for IHCmandates the use of tissue controls processed in a similar way.Microwave procedures require approved laboratory micro-waves and, because of the release of formalin vapors, mustbe performed in a fume hood.

Decalcification

Decalcification with weak acids does not seem to interfere sig-nificantly with IHC for most antigens, provided the tissueswere well fixed in formalin;284 however, decalcification withstrong acid solutions has a negative effect on immunoreactiv-ity, at least for some antigens.7,9,96,232 In a study of melanocyticmarkers in canine tissues, decalcification for less than a weekwith formic acid did not significantly reduce immunoreactivityfor Melan A, PNL2, or tyrosinase, whereas the use of HCl inthe decalcifying solution reduced the immunoreactivity forMelan A and PNL2 after only 1 day of treatment with completeloss of immunoreactivity after 1 week.295 In that study,tyrosinase was more resistant than the other 2 markers to thedeleterious effects of HCl decalcification. The antigenicity ofintermediate filaments appears to be more resistant to decalci-fication effects than other antigens.149 Antigen retrieval in aboric acid solution at 60!C seems to improve the immunoreac-tivity of some antigens in decalcified tissues.414 In summary,weak (eg, formic) acid decalcifying solutions diluted informalin are recommended for IHC. Due to the potential neg-ative effects of decalcifying solutions, the IHC report shouldindicate the type, if any, of decalcification.

Tissue Processing and Incubation Buffers

Although fixation is paramount in the outcome of the antigen-antibody reaction, the incubation buffer and tissue-processingsolutions can also alter antigenicity.89,143 The combination ofcross-linking fixatives with heat and the nonpolar solvents usedin paraffin embedding is thought to modify the antigen confor-mation so that specific epitopes may not be recognized byantibodies that would recognize those epitopes in frozensections. Thus, fixation may not be the sole factor in failureto detect an antigen.89,113,129 There is also evidence for a cumu-lative effect of fixation and tissue-processing factors in the

failure of antigen recognition in FFPE from a study of Ki67 andproliferating cell nuclear antigen (PCNA) immunoreactivity informaldehyde-fixed tissues processed with alcohol andxylene.77,264 Shifts in the tertiary structure of proteins so thathydrophobic areas are oriented outward and hydrophilicregions inward (the so-called hydrophobic inversion)77 duringdehydration and clearing steps of tissue processing can reduceor abolish antibody binding without antigen retrieval, espe-cially with poorly stabilized (unfixed or suboptimally fixedin formalin) tissues/proteins exposed to a weakly polar ornonpolar solvent.264 This negative effect varies with thedehydrating and clearing agent used.93 There is also increasedbackground reactivity in tissues left in xylene for prolongedperiods during processing.149

Few authors have evaluated the effects of long-term storageof formalin-fixed tissues in other solutions.93,359,363 Formalin-fixed tissues stored in 70% ethanol for several weeks had noapparent loss of immunoreactivity or nucleic acid amplifica-tion.359 Tissues stored in tap water for up to a week had no lossof antigenicity or changes in microscopic appearance.363

The effects of dewaxing with organic solvents on IHC havenot been studied systematically. However, nonorganic paraffinsolvents (eg, dishwashing detergents) have been substituted fororganic solvents with comparable results.52,148

Tissue Microarrays

Tissue microarrays (TMAs), introduced by Kononen et al,197

allow simultaneous examination of hundreds of tissues on asingle microscope slide. A tissue core is transferred from the‘‘donor’’ paraffin block to the ‘‘recipient’’ block, which maycontain up to 1000 cores.344 This technology is used not onlyfor protein detection but also for gene expression.y Tissuemicroarray techniques include multitumor arrays (samplesfrom tumors of multiple histological types), progression arrays(samples of normal tissue and different stages of tumorprogression within a given organ), prevalence arrays (tumorsamples from 1 or several entities without extensive clinico-pathologic information to test biomarkers with possible thera-peutic implications), prognosis/outcome-based arrays (sampleswith clinical follow-up data to evaluate prognostic or predictivebiomarkers), cell line arrays (normal or cancer cell lines grownin culture to determine the specificity of antibodies targeting aprotein), heterogeneity/random tissue/tumor arrays (tumor andnontumor tissues from different sites for monitoring theintratumoral heterogeneity of molecular markers), and cryomi-croarrays (frozen samples that might be more suitable thanformalin-fixed tissues for RNA detection).256,316,324,344,367

The advantages of the TMA method include less reagent con-sumption; decreased technical time; decreased variability ofresults; the possibility of digitizing and quantifying results orinterpretation of results by hierarchical cluster analysis, qualitycontrol, and standardization; evaluation of antibody sensitivity

yReferences 120, 121, 146, 172, 243, 256, 258, 266, 316, 346, 381.




and specificity; and rapid and high-throughput discovery andvalidation of biomarkers.173,258,305,311,358 With an adequate selec-tion of control tissues, fewer than 12 tissue cores in an arraysuffice to evaluate more than 90% of the markers used in diagnos-tic IHC.150 With tissue microarray, the cell expressing a particulargene can be identified, whereas in DNA microarray, the sample isdigested before testing, so cell expression cannot be localized.Although the concept of tissue microarray is simple, this methodhas some disadvantages compared with the classic single sample/microscope slide: preparation (with a commercial manual orautomated array) requires high technical skills and careful plan-ning, sample selection is critical due to its small size and the usualheterogeneity of the sample, and differences in fixation protocolsin archival tissue can influence TMA results.99,172,173,266,358

Sampling of multiple and different areas of the donor blockusing small (0.6-mm) cores can be more representative of thelesion and less likely to damage the block than extracting a sin-gle and larger core.99,324 Results from triplicate TMA cores hadup to 98% concordance with the results from full-block sections,whereas concordance was lower with only 1 or 2 cores.99,119

Nevertheless, intratumor heterogeneity can be a complicatingfactor when assessing biomarkers in TMAs,212 especially whena scoring system (eg, 0, 1þ, 2þ, 3þ for HER2 in breast cancer)for the IHC reaction is used for therapeutic planning.

The current Clinical and Laboratory Standards Institute(CLSI) guidelines for IHC recommend TMAs for internal andexternal quality assurance programs. However, due to varioustechnical issues, the use of TMA for diagnostic purposes is notrecommended.125,149 The amount (diameter and number of cores)of tissue needed to test a given biomarker varies and should bebased on the type of tissue/disease process and biomarkerexamined.149

Multiplex immunostaining (MI) chips can be used to examinemultiple antigens in the same tissue section. MI chip technologydiffers from tissue microarray in that MI permits the analysis ofexpression of as many as 50 antigens in a single specimen,whereas the microarray technology permits the analysis of a sin-gle antigen in many specimens simultaneously.117 The mainproblem in applying this technology in a clinical setting is the het-erogeneity of most tumors and, therefore, the possibility of false-negative results.117

Drying of Paraffin Sections

Paraffin sections dried overnight at temperatures of 60!C orhigher may have reduced immunoreactivity for some anti-gens.93,137,147,412 In another study, immunoreactivity wasreduced when slides were dried at temperatures higher than68!C for more than 16 hours, whereas drying at 58!C for 24hours did not affect immunoreactivity.177

Storage of Paraffin Blocks and Tissue Sections

Paraffin blocks containing formalin-fixed tissues are a power-ful resource for protein and nucleic acid investigations.192 Theyare the archived tissue most suitable to evaluate diseases and

apply the results to targeted medicine.354 It is the authors’ andothers’327 opinion that paraffin blocks remain stable for yearsin terms of antigenicity. However, only controlled studies onthe effect of nucleic acid detection in archival paraffin blockshave been published.133 Any deleterious effects of prolongedparaffin block storage on tissue antigenicity could differ amongantigens and should be considered if IHC results on old paraffinblocks are inconsistent or unexpected.

Storage of unstained paraffin control tissue sections increasesefficiency but may adversely affect immunoreactivity (tissue sec-tion aging/slide oxidation/biomolecule degradation).93,127,149

These differences are epitope specific rather than protein targetspecific.149 Both light and temperature contribute to the loss ofantigenicity that occurs with photo-oxidation of tissue sec-tions.30,304 Tissue section aging is a rather common problem withnuclear antigens (eg, Ki67, ER, p53);z however, in a study of ani-mal tissues, cytoplasmic membrane antigens were more sensitivethan nuclear antigens to degradation from photo-oxidation and/orambient temperature.304 Immunoreactivity can also decreasewhen tissues are suboptimally dehydrated (retained endogenouswater) during paraffin embedding or with paraffin sections storedunder high humidity.420 To reduce antigen degradation due tophoto-oxidation, paraffin sections should be used within 1 weekof sectioning or at least should be stored in an airtight containerin the dark.149 Keeping the paraffin sections in the dark underrefrigeration also diminishes the loss of antigenicity.304

In summary, all steps in tissue processing, from acquisitionto storage, can affect the quality of the IHC test.376 When thereis decreased intensity or loss of reaction in stored control tissuesections, the IHC test should be repeated with a different storedcontrol tissue section plus a freshly cut control tissue section. Ifthe change is limited to stored sections, any unused storedcontrol slides should be discarded.

Analytical Phase of IHC

Antigen Retrieval

Fixation and tissue processing modify the 3-dimensionalstructure of proteins, which can render antigens undetectableby specific antibodies because the immunologic reactiondepends on the conformation of the antigen.143 The purposeof antigen retrieval (AR) procedures is to reverse the changesproduced by fixation. AR is particularly important for tissuesfixed in cross-linking fixatives. Approximately 85% of anti-gens fixed in formalin require some type of AR to optimize theimmunoreaction.287 The need for AR and choice of methoddepend on the targeted antigen and the type of antibody;393

AR is more commonly necessary with MAbs than withPAbs.287 The 2 most common AR procedures in IHC areenzymatic and heat-based retrieval. Although AR facilitatesdetection of Ags, background staining or antigen detection in

zReferences 26, 81, 133, 169, 219, 233, 260, 281, 303, 397, 406.




unusual locations is not uncommon with harsh AR methods andcan preclude diagnostic interpretation (Fig. 7).

Antigen Retrieval With Enzymes. Protease-induced epitoperetrieval (PIER), introduced in the mid-1970s,161,162 was themost common AR method before the advent of heat-basedAR in the 1990s. Commonly used enzymes include trypsin,proteinase K, pronase, ficin, and pepsin.20,170,236,263,273 Themechanism of PIER is probably protein digestion, but this clea-vage is nonspecific and some antigens may be negativelyaffected.390 The effect of PIER depends on the concentrationand type of enzyme, incubation parameters (time, temperature,and pH), and the duration of fixation.20,236,273 The enzymedigestion time is proportional to the fixation time.390 It ispractical to optimize a few enzymes for laboratory use. A com-mercially available ready-to-use solution of proteinase K hasgood activity at room temperature, so can be used withautomatic stainers. The disadvantages of PIER are the low num-ber of antigens for which it is the optimal AR method and therisk of altering tissue morphology or destroying epitopes.263,390

Interestingly, proteinase treatment has facilitated IHC detectionof nucleolar proteins present in high concentration butundetectable without AR, whereas nucleolar proteins at lowconcentration can be detected without enzymatic treatment.364

Heat-Induced Epitope Retrieval. Heat-induced epitope retrieval(HIER) has revolutionized the IHC detection of antigens fixedin cross-linking fixatives. In addition, HIER is used forextraction of molecules (eg, nucleic acids, proteins) fromformalin-fixed, paraffin-embedded tissues.332 HIER was intro-duced by Shi et al.337 based on a concept developed byFraenkel-Conrat et al110–112 that the chemical reactionsbetween proteins and formalin could be reversed (Fig. 8), atleast in part, by high-temperature heating or strong alkalinehydrolysis. Although HIER denatures formalin-fixed tissues,it paradoxically restores their immunoreactivity.348 Themechanism involves the dissociation of irrelevant proteinsfrom target peptides.39 The secondary and tertiary structureof proteins is probably modified (denatured) during HIER;however, that should not affect the immunoreactivity of mostantigens because IHC antibodies require only an intact primary(linear) protein structure.39,347 Likewise, some comformationalepitopes become denatured after HIER and will not bindspecific antibodies.421 Heating may unmask epitopes by hydro-lysis of methylene cross-links.135,390,422,423 Rupture of cross-links resets the charged status of proteins and their hydrophobiccharacteristics.421 AR also acts by other mechanisms because itenhances immunoreactivity of tissues fixed in ethanol, whichdoes not produce cross-links.144 Even in unfixed tissues, anti-gens can be unmasked by HIER, perhaps because steric barriersproduced by the antigen itself had precluded antibody access totargeted intramolecular epitopes.180

Other hypotheses to explain HIER are extraction of diffusibleblocking proteins; precipitation of proteins; rehydration of thetissue section, allowing better antibody penetration; and heatmobilization of trace paraffin.349 Tissue-bound calcium ions

may mask some antigens during fixation. Calcium chelating sub-stances (eg, EDTA) are sometimes more effective than citratebuffer in AR.247,248,271,415 Calcium-induced changes in proteinconformation may augment the immunodetection of some anti-gens,399 but for many antigens, calcium-induced effects onimmunoreactivity cannot be documented.331,333 Restoring thenative electrostatic charges modified during formalin fixationhas also been considered an AR mechanism.34,36

Equipment used for HIER includes decloakers (commercialpressure cookers with electronic controls for temperature andtime), vegetable steamers, water baths, microwave ovens, orpressure cookers.13,88,182,271,337 The relationship betweentemperature and exposure time is inverse: the higher thetemperature, the shorter the time needed to achieve results.Heating at high temperature (100!C) for a short duration (10minutes) is better than heating at a lower temperature for a lon-ger time.143 In most instances, the source of heat is not criticalto the outcome but a matter of convenience. Satisfactory resultscan be obtained using a steamer (90!C–95!C) for 20 minutesfor most antigens, although a decloaker eliminates theproblems of irregular heating or temperature variation with asteamer or microwave oven.

A universal antigen retrieval solution is not available.143,167

Thus, several HIER solutions made of different buffers (eg,citrate, tris, Tris-HCl) and pH (3–10) have been used. Someantigens can be retrieved with low pH solutions, others onlywith high pH solutions, and a third group with solutions of awide pH range.332,336 The importance of the chemistry of theretrieval solution, particularly the buffer, is unclear; however,for most antigens, HIER with 0.01 M sodium citrate buffer(pH 6.0) produces satisfactory results with good cell morphol-ogy when compared with buffers with higher pH or solutionscontaining EDTA.19,87,143 However, use of different buffersmay be required to optimize antigen recovery. A low pH buffer(acetate, pH 1.0–2.0) appears especially useful for nuclearantigens. As a rule, high pH and EDTA-containing buffersreach their optimal treatment effect faster than do lower pHcitrate-containing buffers.199 Sometimes multiple AR methods(enzyme-HIER; HIER-HIER) are needed to optimize theimmunodetection of antigens (Fig. 9).97,143,179,340,388 TripleAR was beneficial to demonstrate amyloid b.179

The degree of fixation can dramatically modify the responseof antigens to AR. Unfixed proteins are denatured at tempera-tures of 70!C to 90!C, whereas formaldehyde-fixed proteinsare resistant at the same temperatures.231 This could explainwhy the immunoreactivity of partially fixed tissue can beheterogeneous, despite the supposed even distribution of theantigen.19 In other words, formaldehyde fixation may protectagainst denaturation during AR, although this has been dis-puted, at least when using an autoclave reaching 120!C.39,348

For practical purposes, protein denaturation during AR doesnot have a negative impact on the immunoreactivity of peptides(Fig. 10).

The variability in both the kinetics of fixation and AR fordifferent epitopes suggests that different degrees of cross-linking may occur depending on the peptide amino acid




Figure 6. Effects of fixation on small peptides and cell membrane antigen stability. Formalin fixation stabilizes small peptides and cell membrane anti-gens, preventing leakage or diffusion, even after sectioning. With acetone fixation, small peptides leak through the permeabilized cell membrane. For-malin fixation stabilizes membrane antigens in cryostat sections, but small peptides leak away. Used with permission and modified from Floyd and vander Loos.106 Figure 7. Effects of antigen retrieval (AR) on immunohistochemistry (IHC). Esophagus; dog. (a) No AR. (b) Proteinase K (PK)AR. (c) Heat-induced epitope retrieval (HIER) with citrate, pH 6.0. With no AR, myoglobin expression is observed as expected in skeletal muscle




composition.39 The possibility of unexpected immunoreactiv-ity should always be considered when using HIER, particularlywith low pH buffers.19 When practical, the immunoreactivityof fresh-frozen and routinely processed paraffin tissue sectionsshould be compared331 because not all antigens benefit fromAR, even after prolonged formalin fixation.349

Miscellaneous Antigen Retrieval Methods. Pretreatment withconcentrated formic acid improves the signal in some IHCtests.189 Other AR methods include the use of strong alkalinesolution, urea, borohydride, and a solution of sucrose or acid(eg, hydrochloric acid).330,334 Citraconic acid with heat hasbeen used as AR to reverse formalin-induced cross-linking,presumably by hydrolysis.249,253 This method has beensuccessfully applied to nervous tissue fixed more than 5 years.4

The proposed mechanism of citraconic anhydride (citraconicacid) AR is converting the cross-links in the sample to pro-tected amines and liberating formaldehyde quickly to avoidreforming adducts; then citraconic acid liberates amines viahydrolysis.253

Detergents, although not strictly speaking an AR compo-nent, facilitate Ag-Ab binding by solubilizing membraneproteins.303 Detergents form mixed micelles with lipids as wellas micelles that contain proteins (also known as surfactants),thereby decreasing the surface tension of water. They are usu-ally incorporated into the dilution/rinse buffers. Examples ofdetergents in IHC include ionic detergents, bile acid salts, andnonionic and zwitterionic types (eg, Triton R-X100, BRIJR,Tween 20, saponin). The addition of sodium dodecyl sulfate(SDS) to the AR buffer and HIER at relatively low temperature(97!C) appears to improve the signal of many markers.366

All-in-One Epitope Retrieval Buffers. The classic histologic proce-dure calls for dewaxing paraffin sections using organic solvents(eg, xylene) followed by dehydration in alcohols and hydrationbefore AR. The so-called 3-in-1 reagents can perform aqueousdeparaffination, rehydration, and AR.149 Their use in IHC isrelatively new, so comparison with traditional means of depar-affination, rehydration, and AR is required before using them ina diagnostic setting. Commercially available all-in-one epitoperetrieval buffers can reduce processing time by melting theparaffin in tissue sections during HIER.148 The advantage ofthese solutions is obvious, but reported instances of incompleteparaffin removal could affect the outcome of the IHC.114 Thisis more commonly observed with solutions based on surfac-tants and wetting agents, which tend to incompletely solubilizethe molten paraffin. The addition of a water-soluble organic

agent appears to solve this problem without negatively affect-ing the IHC reaction.114

Antigens and Antibodies: The Specificity Issue

Immunohistochemistry involves antibody recognition andbinding to an epitope on the target antigen.149 The specificityof the reaction largely depends on qualities of the primaryantibody and the ability of the antigen (epitope) to bind it. Themost commonly used immunoglobulin (Ig) is IgG; IgM is usedless commonly.284

Antigens. Antigens can be as small as a monosaccharide;epitopes consist of 5 or 6 amino acid residues.149 Antigens canbe multivalent with multiple identical epitopes (homopoly-meric) or multiple distinct epitopes (heteropolymeric).213 Asingle gene can generate several different antigen isoforms via2 principal mechanisms. Alternative splicing of the primarygene transcript can produce multiple different maturetranscripts, each of which codes for a slightly differentprotein.44,328 Many proteins also undergo posttranslationalmodifications, such as glycosylation, phosphorylation, andproteolytic processing, which add further complexity.237 As aresult, 1 gene can generate numerous protein (antigen) iso-forms, and this repertoire can change with time (eg, tenascinand hemoglobin isoforms change from fetal development toadulthood).237

Two broad groups of immunogens are used to produceantibodies: synthetic peptides and purified protein prepara-tions.237 The known amino acid sequence of synthetic peptidesfacilitates IHC interpretation, both with respect to the isoformsof the target protein and any cross-reactivity with similar pep-tide sequences in other proteins. A potential disadvantage tousing synthetic peptides is that an isolated synthetic peptidesequence may lack the normal 3-dimensional structure of thenative protein. In addition, other proteins can be intimatelyassociated with the protein of interest in vivo. Either factor canmask target epitopes, preventing their detection by antibodiesraised to synthetic peptides and therefore yielding false-negative results. Also, crucial in vivo posttranslational modifi-cations of the native antigen are absent in synthetic pep-tides.220,221 The presence of the synthetic peptide sequence inunrelated proteins could produce immunologically specificAg-Ab binding (molecular mimicry) despite the absence of thetarget antigen.29,72,165 Use of purified proteins as immunogensavoids many of the problems of synthetic peptides.237 How-ever, protein purification to homogeneity from cells or tissuescan be technically difficult, and contaminating proteins may be

Figure 7. (continued) (solid circle) with no labeling of mucosal epithelium (arrowhead), plasma (V), or smooth muscle (asterisk). With PK AR,background reactivity develops in mucosal epithelium, plasma, and smooth muscle. Nonspecific labeling with HIER, in contrast to that with PK,has a predominantly nuclear pattern in epithelium and smooth muscle (inset). Immunoperoxidase-DAB for myoglobin, hematoxylin counterstain.Figure 8. Conformational changes in protein structure from cross-linking fixatives. Modification of epitope (colored segments) presentation couldpreclude access by specific antibodies. Fixation-induced conformational changes can be reversed by HIER. Figure 9. Antigen retrieval. Kidney; dog.Collagen type IV was demonstrated by sequential double AR with HIER (citrate) and pepsin. No reactivity with pepsin AR alone (lower inset) or HIERalone (upper inset). Immunoperoxidase-DAB, hematoxylin counterstain.




Figure 10. Loss of immunoreactivity after formalin fixation with recovery by antigen retrieval (AR). (a) Putative structural changes in nativeproteins. (b) Corresponding changes to epitopes in the in vitro peptide assay. Used with permission from Sompuran et al.347




more antigenic than the protein of interest, producing a dispro-portionate and unwanted immunogenic response. Anotherproblem with purified proteins arises if the targeted antigenincludes highly immunogenic epitopes that are not specific tothe antigen of interest. This pitfall is common when similarposttranslational modifications occur among antigens that dif-fer markedly in amino acid sequence. Unfortunately, the sourceand details of antigen purification or the isoforms of the tar-geted protein recognized by an antibody preparation are seldomreported for commercial primary antibodies.237 Other factorsaffecting immunogenicity include size, conformation, and elec-trical charge of the antigen; use of adjuvants; and dose androute of administration of the antigen.

Antibody Structure. Immunoglobulins are ‘‘Y’’ shaped andconsist of 2 identical light chains and 2 identical heavy chains(Fig. 1). The heavy chains determine the antibody class. Thetail of the Y, called Fc (Fragment, crystallizable), is composedof 2 heavy chains on the C-terminal side.46 Each end of theforked portion of the Y is called the Fab (Fragment, antigenbinding) region. The light chains of most vertebrate antibodieshave 2 distinct forms, k and l. In any immunoglobulin mole-cule, both light chains and both heavy chains are of the sametype. The light chains are divided into the C-terminal half,which is constant and called CL (Constant: Light chain), andthe N-terminal half, which has abundant sequence variabilityand is called the VL (Variable: Light chain) region. The Fab(antigen-binding) region of the immunoglobulin has variableand constant segments of the heavy and light chains. The Fcportion determines biological functions and permits antibodybinding to other antibodies, complement, and immune cellswith Fc receptors. This portion of the Ig is needed for multistepIHC techniques284 and is largely responsible for nonspecificbackground reactivity due to nonimmune adherence of antibo-dies (Abs) to the tissue section. Background can be diminishedby the use of only Fab or F(ab)2 portions of the Ig molecule forIHC; however, without the Fc portion, antibody binding tosolid substrates, such as tissues, is less stable. The specificbinding of an antibody to an antigen occurs via hypervariableregions of both heavy and light chains of the amino termi-nus.284 The antigen-binding site of an Ab is called the paratope.Epitopes, usually 5 to 21 amino acids long, are the regions of anantigen (Ag) that bind to antibodies.141 In an immunologicreaction, one of the most important criteria for antibody bind-ing is the tertiary structure of the epitope (ie, the way in whichpeptide chains of a protein are folded or interact with adjacentpeptides). The paratope interacts with the tertiary structure ofthe epitope through a series of noncovalent bonding (seebelow). The more bonding, the greater the affinity andavidity of the antibody. IgG antibodies are bivalent (have 2identical arms used in antigen recognition). This is a key com-ponent of multiple-layer immunohistochemical methods. Otherimmunoglobulins, except secretory IgA, which is tetravalent,and IgM, which is decavalent, are also divalent.

Epitopes are classified as linear or conformational.39 Linearepitopes are a group of 5 to 7 contiguous amino acids.

Conformational (discontinuous) epitopes, the typical form,consist of small groups of amino acids brought together by con-formational folding or binding.39 Although most epitopesinvolved in immune responses are believed to be conforma-tional, data support the concept that antibodies used in FFPEtissues recognize mainly, or exclusively, linear epitopes.39,347

The Nature of Antigen-Antibody Interactions. From a biochemicalpoint of view, Ag-Ab interactions are somewhat unusual.284 Thebonds are weak (mostly hydrophobic, van der Waals, and elec-trostatic) and not covalent. Hydrophobic bonds develop betweenmacromolecules (interatomic or intermolecular) with surfacetensions lower than that of water. Hydrophobic interactions areimparted mainly through the side chain amino acids phenylala-nine, tyrosine, and tryptophan.31 By their lower attraction towater molecules, these amino acids tend to link with oneanother. Electrostatic (Coulombic) interactions (also calledionic bonds) are caused by attractive forces between 1 or moreionized sides of the antigen and oppositely charged ions on theantibody-active site. These typically are the carboxyl and theamino groups of polar amino acids of the Ag and Ab molecules.van der Waals forces are weak electrostatic interactions betweendipolar molecules or atoms.2 van der Waals forces and electro-static attractions are maximal at the shortest distances. There-fore, precise juxtaposition of oppositely charged ions onepitopes and paratopes favors strong electrostatic bonding.2

Hydrogen bonds are the result of dipole interactions betweenOH and C¼O, NH and C¼O, and NH and OH groups with bind-ing energy of the same order of magnitude as that of van derWaals and electrostatic interactions. Their importance in Ag-Ab interactions is diminished by the necessity of a precise fitbetween molecules. Although there are situations with only 1type of interaction, for most polysaccharide, glycoprotein, andpolypeptide Ags, the Ag-Ab bond is a combination of van derWaals forces and electrostatic interactions.2 Most proteinantigens are multivalent, and each valency site is generally anantigenic determinant (epitope) with a completely different con-figuration from the other sites; a monoclonal antibody (MAb)can react with only 1 valency site of such an Ag.391 Table 3 liststhe major factors that affect antigen-antibody binding.

The antibody-antigen bond is reversible, and its strengthcan be quantified.149 Affinity is a thermodynamic expressionof the binding strength of an antibody (paratope) to an anti-genic determinant (epitope).213 The affinity constant (Ka) rep-resents the amount of antibody-antigen complex formed atequilibrium.

Ab Ag Ab ~ Ag x caloriesaK

Ka ¼½Ab % Ag&½Ag& ' ½Ab&

where Ab*Ag are antigen-antibody complexes and thebracketed terms indicate molar concentrations. Ag-Ab affinityconstants range from below 105 L/mol to 1012 L/mol (an




Ag-Ab complex with a Ka of 1012 L/mol has 1000-fold greateraffinity than one with a Ka of 109 L/mol). Affinity can be deter-mined precisely only for monoclonal antibodies (MAbs);213 forpolyclonal antibodies (PAbs), composed of antibodies with dif-ferent affinities, affinity can only be estimated. Anothermethod currently used by antibody manufacturers to calculatethe affinity of antibodies is determining the equilibrium disso-ciation constant (Kd). Most antibodies have Kd values in lowmicromolar (10–6) to nanomolar (10–7 to 10–9); very high affi-nity antibodies have picomolar (10–12) values. In other words, ahigh Ka value (eg, 1012) will correspond with a very low Kd

value (eg, 10–12). New high-throughput technologies such asmicroarray-based kinetic constant assays201,202 allow the Kd

calculation for a large number of substances simultaneously.The affinity of an antibody for an antigen influences thesensitivity and specificity of an immunologic reaction.149

Intrinsic affinity is determined by the sequence of amino acidsof an epitope, which in turn determines the specificity of anantibody.149 In general, high-affinity antibodies bind moreantigen in less time than low-affinity antibodies, so the higherthe affinity, the more dilute the antibody solution can be.113,149

Avidity, or functional combined bond affinity, is a measureof the overall binding intensity between antibodies and amultivalent antigen.149,213 Avidity is the result of the affinityor affinities of the antibody for the epitope(s), the number ofantibody binding sites, and the geometry of the antigen-antibody complexes.213 Thus, an antibody of IgM (decavalent)has a higher avidity (although typically lower affinity) than anantibody of IgG (bivalent) type.149,213 High-avidity antibodieshave multiple sites for secondary reagent binding, which isparamount in IHC.213 The lower the affinity, the longer the reac-tion time; prolonged incubations may increase the nonspecificbackground.149 The functional affinity of a PAb may be higherthan that of a MAb due to its capacity to bind multiple epitopeson a single antigen.149 To increase the functional affinity ofMAbs, cocktails of antibody clones targeting the same proteinare sometimes used.149

Monoclonal and Polyclonal Antibodies

Polyclonal Antibodies. Polyclonal antibodies are produced byimmunizing rabbits and various other species (mouse, goat,donkey, horse, hamster, sheep, guinea pig, rat, chicken) withpurified antigen. Chickens transfer abundant IgY (IgG) intoegg yolk, which makes harvesting antibodies from eggs moreconvenient than the typical bleeding procedure used in mam-malian species.46,213 Polyclonal antibodies have higher affinity

and broader reactivity but lower specificity in comparison tomonoclonal antibodies.141 Polyclonal antisera include severaldifferent antibodies to the target protein plus, if not affinity pur-ified, irrelevant antibodies in concentration up to 10 mg/ml.89,237 Polyclonal antibodies are more likely than MAbs toidentify multiple isoforms (epitopes) of the target protein.However, a polyclonal preparation that has not been affinitypurified may be heterogeneous due to the combined presenceof high-affinity antibodies and weakly immunogenic epi-topes.237 Variations in antibody titer and quality, dependingon the animal immunized, also fluctuate from lot to lot.255 The‘‘immunologic promiscuity’’ of PAbs, which can amplify anti-gen detection by recognition of multiple epitopes, can be a dis-advantage: the greater the number of different antibodies to thetarget protein, the greater the likelihood of cross-reactivity withsimilar epitopes in other proteins and, therefore, the likelihoodof false positives.237 Many proteins share immunogenicepitopes. For instance, common immunogenic domains offibronectin III are present in at least 65 unrelated proteins.41

Monoclonal Antibodies. Monoclonal antibodies are produced bythe technique of Kohler and Milstein,196 in which an animal,usually a mouse, is immunized with purified antigen. Thespecific antibody-producing B lymphocytes are then harvestedfrom the spleen and fused with mouse myeloma cells to pro-duce immortal hybrid cells that produce Igs specific for a singleepitope (MAbs). The immortalized hybrid cells form hybrido-mas that are selected for the desired specificity and can bemaintained in cell cultures (highly pure antibody but at lowconcentration) or in the peritoneum of mice (ascitic fluid with10- to 100-fold higher Ab concentration than in cell culture, butwith nonspecific proteins and extraneous endogenous Igs). Oneadvantage of monoclonal over polyclonal antibodies is theirhigher specificity (Table 4). In addition, background reactivitydue to nonspecific Igs is reduced (ascites fluid) or nonexistent(cell culture supernatant). The high specificity does not elimi-nate the possibility of cross-reactivity with other antigensbecause MAbs target epitopes consisting of only a few aminoacids, which can be part of multiple proteins and peptides.254

For instance, an anti–human proinsulin antibody cross-reactswith both insulin and glucagon-secreting cells.23 Because thisbinding is to an identical epitope, the IHC reaction is virtuallyindistinguishable from that with the intended epitope.365 It canbe difficult to determine whether immunoreactivity reflectsshared epitopes (cross-reactivity) or epitopes resulting fromprotein cross-linking during fixation with aldehydes.141

Table 3. Main Forces Involved in Antigen-Antibody Binding.391

Types of Forces Ag-Ab Binding Favored by Ag-Ab Dissociation Favored by

van der Waals High incubation temperature Reduction of surface tension bufferElectrostatic Neutral pH of buffer Extreme pH of buffer

Low ionic strength of buffers High ionic strength bufferLow incubation temperature High incubation temperature




Rabbit Monoclonal Antibodies. The production of rabbit MAbshas been facilitated by the development of antibodylibraries.278,283,307 The advantages of rabbit over mouse MAbsinclude higher affinity (probably a result of high glycosyla-tion),149 suitability for use on mouse tissues without specialprocedures, increased specificity in some cases, and less needfor antigen retrieval methods.57,132,211,313,417 Nevertheless, ina study comparing 10 rabbit MAbs with 10 mouse MAbs tar-geting the same protein in animal tissues, rabbit MAbs weresuperior for some markers, but there was no significant overallimprovement in immunoreactivity.396

Protein A and Protein G. These cell-wall components of Staphy-lococcus aureus (A) and group G (G) streptococcal strains46 areused in IHC for their high affinity for the Fc portion of IgGs.The IgG of many species can be bound to protein A or proteinG, creating a versatile label when conjugated to enzymes orfluorochromes. Protein A/G is a genetically engineered productof Bacillus sp that combines the IgG-binding domains ofprotein A and protein G. Reportedly, it has higher affinity invarious species than protein A or protein G alone.46

Antibodies to Phosphorylated Proteins. These antibodies are spe-cific for a protein activation state, so may indicate a biologicalstate rather than the mere presence of a protein.149,222 However,the production of these antibodies is challenging because thephosphorylation sites are at consensus sequences shared bymany proteins.149

Mouse-on-Mouse IHC. The use of mouse MAb for IHC of mouseor rat tissues results in excessive background labeling due tothe binding of the secondary antibody (anti–mouse immuno-globulins) to endogenous immunoglobulins in the interstitium,plasma, B lymphocytes, and plasma cells.49,116 This excessivebackground labeling has hampered or even precluded IHC with

mouse MAbs in murine tissues; however, commercially avail-able mouse-specific detection systems have eliminated thisproblem. One such system uses blocking steps before and afteradding the primary antibody; another method preincubates theprimary antibody with biotinylated anti–mouse Fab complexes(used as secondary antibody), thereby blocking free bindingsites in the complexed secondary antibodies with normalmouse serum before adding the antibody mix to the tissue sec-tion.116,152,227 Secondary antibodies to mouse IgGs may alsoreact with Igs of other species producing the same type of non-specific background in plasma cells, serum, or any tissue con-taining IgGs. The use of isotype-specific secondary antibodies,because of the low serum concentration of each isotype (20%–25%), is a relatively inexpensive method to minimize this typeof background.49

What Makes an Antibody Good for Immunohistochemistry? Speci-ficity and sensitivity are the most important traits. Specificity isinherent in the primary antibody. Molecular specificity, theselectivity of an antibody for a particular epitope of the targetantigen, depends on its molecular structure.149 The diagnosticspecificity of an antibody against infectious agents is quantifiedas the proportion of samples from known uninfected animalsthat test negative in a given test.419 The diagnostic specificityof an antibody against cellular/tumor markers is the expectedpresence or absence of immunoreactivity in certain cell types,tissues, or tumors (see Controls in Immunohistochemistrysection).369 Analytical sensitivity is determined by the leastamount of antigen needed to produce a positive reaction. Poly-clonal antibodies may be more sensitive than MAbs becausethey bind different epitopes on a single antigen.369 Diagnosticsensitivity, the proportion of known positive samples that testpositive in a given test,374,419 is best established by comparingtest results on FFPE tissue with results using another antibodyvalidated for the same analyte or a non-IHC method, such as

Table 4. Comparison of Polyclonal and Monoclonal Antibodies for Immunohistochemistry.

Polyclonal Ab Monoclonal Ab

Origin Multiple species Mouse, rabbitTotal amount of antibody 5–20 mg/ml 0.05 mg/ml (serum-free medium culture supernatant)

1–10 mg/ml (ascites)Amount of specific antibody 0.05–0.2 mg/ml 0.05 mg/ml (serum-free medium culture supernatant)

0.9–9 mg/ml (ascites)Production Easy and inexpensive More technologically challenging and expensive

Large volume of antibody from the same animal Limitless amount from hybridoma cell linesEpitope recognition Multiple, including multiple isoforms of the same protein One single epitopeSpecificity Low to high HighAffinity Variable (both high and low) HomogeneousAvidity High Lower than polyclonal antibodiesFixation tolerance High Variable to lowFixation effects Multiple SingleMolecular specificity Lower than for monoclonal antibodies High, resulting in less background (background from

mouse antibodies in ascites fluid)Antibody batch heterogeneity Variable Minimal

Limited number of batches Unlimited number of batches




culture or polymerase chain reaction (PCR). Nonspecific bind-ing of antibodies in IHC can be reduced or eliminated withreagents (including the primary antibody) of good quality;therefore, the nonspecific binding blocking step included inmost IHC protocols may be unnecessary in some instances.50

Finally, antibody selection depends on its performance intissue sections (see standardization and validation below). AsKalyuzhny181 wrote, ‘‘Having a good antibody for immunohisto-chemistry is not only about getting a strong staining signal withlow background, but also about knowing the staining makes sensein terms of its histological and physiological relevance.’’

Presentation of Commercial Antibodies

For a well-characterized antibody, the manufacturer’s packageinsert should provide all pertinent information.368 For compa-nies specialized in antisera production, this should include thenature of the antibody (eg, purified, whole serum, supernatant,ascites, immunoglobulin isotype), host (eg, mouse, rabbit) inwhich it was produced, protein concentration, immunogen used(including epitope and molecular weight, if known), speciesreactivity/expected reactivity (eg, human, mouse; others notknown), cellular localization (eg, cytoplasmic, membrane,nuclear), recommended positive tissue controls, applications(eg, immunoprecipitation, Western blotting, enzyme-linkedimmunosorbent assay, immunohistology–formalin/paraffinand frozen), antigen retrieval, suggested antibody dilution, andpertinent references. Manufacturers may add client reports onspecies cross-reactivities. Many catalogs also include imagesof the immunoreaction and Western blot gels with the molecu-lar weight of the targeted epitope(s) and the percent proteinhomology across species. The protein concentration of an anti-body can be used to estimate the working titer; however, this isonly accurate for MAbs in which the specific antibodies consti-tute the bulk of the reagent. For PAbs, protein concentrationdoes not predict performance due to the presence of nonim-mune proteins or antibodies with variable affinity, in additionto specific Igs. The manufacturer should be contacted foradditional information on reactivity under certain conditions(formalin fixation) or species cross-reactivity if not indicatedin the catalog. Data provided by only very few manufacturersin selected antibodies are their affinity constant/dissociationconstant and the possibility of cross-reactivities with other anti-gens (eg, serotypes of viruses, other related viruses or bacteria,cell antigens) that may result from fixation or antigen retrievalprocedures.

How to Find the Antibody That Matches Your Needs. Antibodiesare selected based on consultation with other scientists, publi-cations, and information from manufacturers.47 Useful Internetresources include the Human Protein Atlas (http://www.protei-natlas.org/), which contains information on protein and subcel-lular profiles of numerous human cancers.25,276,378 The Atlaslists more than 17 000 antibodies targeting proteins from morethan 14 000 genes with high-resolution images to depict reac-tivity of the antibodies in tissue sections. Other sites with

information on commercial antibodies are http://www.anti-bodypedia.com, http://www.biocompare.com, http://antibodyr-esource.com, http://www.antibodydirectory.com, and http://www.linscottsdirectory.com. Some provide information aboutIHC cross-reactivity and techniques for FFPE tissues or frozensections for animal species. The South Dakota State Universitydatabase is dedicated to IHC in animal samples (http://www.sdstate.edu/vs/adrdl/database/). The PHL Murine Immu-nohistochemistry Database (http://ncifrederick.cancer.gov/rtp/lasp/phl/immuno/) lists many biomarkers with specific proto-cols for IHC in mouse tissues.

Detection Systems: The Sensitivity Issue

The primary, secondary, or tertiary antibodies of a detectionsystem are labeled with reporter molecules (labels) to allowvisualization of the antigen-antibody reaction. Suitable labelsinclude fluorescent compounds, enzymes, and metals.218,370

The most common labels are enzymes (eg, peroxidase, alkalinephosphatase, glucose oxidase).303 Enzymes in the presence oftheir substrate and a chromogen produce a colored precipitateat the site of the antigen-antibody reaction.284

The detection system is important to maximize sensitivity ofan IHC test and to optimize visibility of the immune reactionwith the fewest steps and in the shortest time.149 In addition, thedetection system must be reproducible, be accurate, and rendera high signal-to-noise ratio. Other factors to consider in selec-tion of a detection system include (1) the expertise/experienceof the technician, (2) type of antigens to be detected (less abun-dant antigens need highly sensitive methods), (3) number oftests (antibodies) available (different antibodies may requiredifferent detection systems), (4) species/tissue idiosyncrasies(eg, amount of endogenous biotin), and (5) budget.293 Last butnot least, the detection system must be compatible with the ani-mal species. A detection system with excellent performance inhuman tissues may not perform well in animal tissues. There isa trend in companies marketing IHC detection systems forveterinary use to optimize their detection kits based on the spe-cies evaluated (eg, PromARK for dogs and cats, food animals,etc), reducing the chances of background or false-positivelabeling. In this article, detection systems are classified asdirect or indirect methods.

Direct Methods. Direct detection methods are a 1-step processusing a primary antibody conjugated (labeled) with reportermolecules,66 such as fluorochromes, enzymes, colloidal gold,or biotin.275 The direct method is quick but lacks sufficient sen-sitivity for the detection of most antigens in routinely processedtissues (Fig. 11).

Indirect Methods. The need for more sensitive antigen detectionprompted Coons et al67 to develop a 2-step method. The firstlayer of antibodies is unlabeled, but the second layer, raisedagainst the primary antibody, is labeled (Fig. 11).275 The sensi-tivity is higher than with a direct method because (1) the unla-beled primary antibody retains full avidity with stronger Ag




Figure 11. Direct and indirect immunohistochemistry (IHC) methods. Figure 12. Avidin-biotin complex (ABC) method. Figure 13. Labeledstreptavidin-biotin (LSAB) method. Figure 14. Peroxidase-antiperoxidase (PAP). Figure 15. Polymer 1-step method. Figure 16. Tyramide-biotin immunoperoxidase method. Figure 17. Nonspecific labeling with inappropriate detection system. Two detection systems were used:LSABþ (a, b), a universal detection system labeling rabbit, mouse, and goat primary antibodies; EnVisionþ (ENVþ) (c, d), a polymer detectionsystem labeling only mouse primary antibodies. Upper panel: Carcinoma, skin; dog. Minimum background with LSABþ (a) and none withEnVisionþ (c). Lower panel: Mammary gland; goat. Extensive background with LSABþ (a) due to binding to endogenous goat immunoglobulins.




binding, and (2) the number of labels (eg, peroxidase) permolecule of primary antibody is higher, increasing the reactionintensity. Indirect methods can detect smaller amounts of anti-gen with less primary antibody because at least 2 labeledimmunoglobulins can bind each primary antibody molecule.Indirect methods are also more convenient than the directmethod because the same secondary antibody can be used todetect different primary antibodies, provided that the latter areraised in the same species.275

Avidin-Biotin Methods

Avidin is a large glycoprotein, extracted from egg white,with 4 binding sites per molecule and high affinity forbiotin. Biotin has 1 binding site for avidin and can beattached through other sites to an antibody (biotinylatedantibody) or another macromolecule, such as an enzyme,fluorochrome, or other label.275 The increased sensitivityof avidin-biotin methods reflects the larger number of biotinmolecules (reporter molecules) that can be attached to a pri-mary antibody.134,159,160

One of the most common avidin-biotin methods is theavidin-biotin complex (ABC) method, in which the secondaryantibody is biotinylated, and the third reagent is a complex ofavidin mixed with biotin that is linked with an appropriate label(eg, enzyme) (Fig. 12). The avidin and labeled biotin are incu-bated for about 30 minutes before application, resulting in theformation of a large complex with numerous molecules oflabel. The proportion of avidin to labeled biotin in the thirdreagent must leave some sites free to bind biotin on the second-ary antibody.275 Another commonly used avidin-biotin methodis the labeled avidin-biotin (LAB) or labeled streptavidin-biotin (LSAB) (Fig. 13). This method uses a biotinylatedsecondary antibody and a third reagent of peroxidase (or alka-line phosphatase)–labeled avidin. Its sensitivity is higher thanthat of the standard ABC method.90

A disadvantage of any avidin-biotin system is the possibilityof high background. Avidin can produce background by bind-ing lectins and negatively charged tissue components throughits carbohydrate groups or through electrostatic bindingbecause its isoelectric point is 10. This background can bediminished by substituting streptavidin for avidin. Streptavidin,produced by the bacterium Streptomyces avidinii, has a neutralisoelectric point, resulting in marked reduction of electrostaticinteractions with tissue. In addition, because streptavidin doesnot bind lectins, background labeling is less likely but stillpossible due to endogenous biotin. Endogenous biotin activityor tissue affinity for avidin, known as endogenous avidin-biotinactivity (EABA), is particularly common in tissues that are richin biotin, such as the liver, brown fat, adrenal cortex andkidney. Mast cells also have EABA.138 EABA is accentuated

by HIER but also develops in tissues subjected to other typesof AR.56,187,239,257,275 EABA is typically in the cytoplasm buthas been reported in the nucleus.250,257,342,428 Commerciallyavailable EABA blocking reagents (pure avidin and biotinsolutions) are expensive, so many laboratories use ‘‘home-made’’ solutions with egg white as a source of avidin and 5%powdered milk as a source of biotin.12,84,175,239,285,418

Non–Avidin-Biotin Methods

Peroxidase-Antiperoxidase (PAP) Method. This indirect methodconsists of 3 layers.357 The third layer, which for a rabbitprimary antibody is a rabbit antiperoxidase, is coupled withperoxidase in proportions to form a stable complex (peroxi-dase-antiperoxidase) of 2 rabbit IgG molecules with 3 peroxi-dase molecules, one of which they share (Fig. 14).275 The firstand third layers are bound by a bridge layer of immunoglobulins(in this example, anti–rabbit Ig). The key is to add the secondary(bridge) antibody in excess so as to bind the primary antibodythrough one binding site and the PAP complex through the other.The peroxidase-antiperoxidase (PAP) method is more laborious,but has 100- to 1000-fold higher sensitivity than the 2-stepindirect method. PAP reagents are available for use with goat,mouse, rabbit, rat, and human primary antibodies.370 AlthoughPAP IHC was popular before the advent of avidin-biotin meth-ods, its lower sensitivity limits its use today.

Polymeric Labeling 2-Step Method. This method uses a compactpolymer to which 4 to 70 molecules of enzyme (peroxidaseor alkaline phosphatase) plus 1 to 10 molecules of the second-ary antibody are attached (Table 5).149,411 Its advantages are(1) simplicity compared with the 3-step methods, (2) equalor higher sensitivity than ABC or LSAB methods, and (3)lack of background due to endogenous biotin or avi-din.269,318,335,370,398 However, this method is usually moreexpensive than ABC or LSAB methods (Fig. 15). Numerouscompanies have commercialized polymer-based detection sys-tems (eg, EnVision, PowerVision, ImmPRESS, MACH 4M),which are the method of choice in many laboratories. The sen-sitivity can be increased with a 3-step method, in which abridge antibody links the primary antibody and the polymercomplex. Newer detection kits have replaced dextran or othermacromolecules with smaller polymers (micropolymers) toincrease access to the target antigen, resulting in higher sensi-tivity, lower background, and reduced nonspecific binding.149

Catalyzed Signal Amplification. This method is based on the abil-ity of tyramide to adhere to a solid substrate (eg, tissue section)following oxidation/radicalization.131 The procedure, adaptedfor IHC,3 is based on the deposition, catalyzed by horseradishperoxidase (HRP), of biotinylated or FITC-conjugated tyra-mide at the location of the antigen-antibody reaction. Free

Figure 17. (continued) No background when using EnVisionþ (c). (b, d) Negative reagent controls. Immunoperoxidase-DAB for cytokeratinsusing a mouse monoclonal antibody, hematoxylin counterstain. Figure 18. Double immunolabeling using 2 mouse monoclonal antibodies withsame class isotype immunoglobulin. Figures 11 to 16 and 18 are modified from Ramos-Vara,284 with permission from Veterinary Pathology.




radicals formed during the HRP-tyramide reaction bind cova-lently to electron-rich amino acids (eg, tyrosine) of nearby pro-teins.48 The reactive intermediates are so short-lived thatbiotinylated tyramide is deposited only at or near its site of pro-duction.143 The biotin conjugated to the tyramide subsequentlycomplexes with avidin conjugated to HRP.143 This method iscomplex and laborious because it involves an initial avidin-biotin procedure followed by the tyramide reaction (Fig. 16).However, its sensitivity is 5- to 10-fold higher than that of theregular avidin-biotin method;350 others claim an even greaterincrease in sensitivity.234 This method is suitable for antigenspresent in very low amounts. However, background can be aproblem, particularly when using HIER,140 so prolonged treat-ment to quench endogenous peroxidase or EABA is usuallynecessary. Modifications using fluoresceinated tyramide resultin marked reduction or ablation of background from endogen-ous biotin.139,140,259,389

Immuno-Rolling Circle Amplification. Rolling circle amplification(RCA) increases the signal of the immunologic reaction with-out increasing the noise (background) that can occur with thetyramide amplification method. Rolling circle amplificationis a 2-part, surface-anchored DNA replication used to visualizeantigens (immunoRCA). The first part is an immunologic

reaction (antigen-antibody binding); the second part is anisothermal nucleic acid amplification using a circularizedoligonucleotide primer.216,325,436 The primer is coupled to theantibody, so in the presence of circular DNA, DNA polymer-ase, and nucleotides, the rolling circle reaction results in a DNAmolecule consisting of multiple copies of the circular DNAsequence that remain attached to the antibody. The amplifiedDNA can be detected by hybridization with labeled comple-mentary oligonucleotide probes.325 The main differencesbetween RCA and PCR is that the former can amplify nucleicacid segments in either linear or geometric kinetics under iso-thermal conditions, and the product of amplification remainsconnected to the target molecule.216,325 The linear mode ofRCA can generate 105-fold signal amplification during a briefenzymatic reaction. ImmunoRCA is sensitive enough to detecta single antigen-antibody complex.325

Polyvalent Detection Systems. Many manufacturers offer polyva-lent (sometimes called universal) detection systems. Theydiffer from 1-species detection systems in that the secondaryreagent is a cocktail of antibodies against immunoglobulinsfrom different species, allowing 1 secondary reagent to be usedfor both polyclonal (eg, rabbit and goat) and monoclonal (eg,mouse) antibodies. These systems reduce the complexity ofIHC; however, due to the ‘‘universal’’ recognition by thepolyvalent antibodies, awareness of potential species incom-patibilities is critical (Fig. 17).

Increasing the Sensitivity of Antigen Detection. The moresophisticated and highly sensitive detection methods can beprohibitively expensive. Therefore, standard methods (eg,PAP, ABC, LSAB) have been modified by combining meth-ods, repeating steps of the immune reaction, increasing theincubation time of the primary antibody, or enhancing theintensity of the chromogen precipitate.§

Detection of Multiple Antigens in a Tissue Section. Multipleimmunolabeling is used to localize different antigens (eg, cellmarkers plus infectious organisms) in the same section. Theavailability of various detection systems (PAP, ABC,polymer-based methods, commercial dual-labeling kits),enzymes, and chromogens with different-colored reactionsmakes multiple immunolabeling feasible. A potential pitfallis false-positive labeling due to cross-reactivity among differ-ent components of the reaction. This requires careful planningand the use of multiple controls. Double immunolabeling meth-ods can be simultaneous (parallel) or sequential.382 Simulta-neous double immunolabeling is performed with primaryantibodies made in different species; both primary antibodiesare added simultaneously, followed by a mixture of differentenzyme-conjugated secondary antibodies and subsequentlydeveloped with the appropriate chromogen/substrate solu-tions.149 As a rule, if the primary antibodies are from the samespecies, sequential dual labeling is necessary. The risk with

§References 60, 78, 224, 225, 275, 315, 370.

Table 5. Standard Immunohistochemical Protocol Using a 2-StepPolymer-Based Detection System.a

1. Deparaffinize slides and bring them to deionized water2. Antigen retrieval (eg, enzyme, HIER) procedure, if needed3. Rinse in deionized water4. Block endogenous enzyme activities (eg, peroxidase)5. Rinse sections and bring them to buffer6. Nonspecific staining blocking7. Remove excess of blocking solution (do not rinse)8. Incubate section with primary antiserumb

9. Rinse slides with buffer10. Incubate with secondary reagent (immunoglobulin anti–primary

antibody species)11. Rinse slides with buffer12. Incubate slides with tertiary reagent (enzyme-immunoglobulin-

polymer complex)13. Rinse slides with buffer14. Detection of the immune reaction with developing reagents (eg,

DAB and H2O2 for peroxidase)15. Rinse sections with deionized water16. Counterstain with hematoxylinc

17. Dehydrate and coverslipd

HIER, heat-induced antigen retrieval.aThe best incubation times for the primary antibody and other reagentsshould be based on information provided by manufacturers of variousreagents and by personal experience.bIncubation of the primary antibody can be done at room temperature, 37!C,or 4!C. The best incubation temperature should be determined by trial anderror. The incubation chamber should have enough moisture to avoiddessication of the slides.cSelect carefully the type of counterstain based on the chromogen used andlocation of the antigen.dUse water-based coverslipping media when the precipitate of the enzymaticreaction is sensitive to organic solvents.




sequential dual labeling is that the second layer of antibodies(intended for the second antigen) can also bind the first antigenthrough the primary antibody (Fig. 18). Therefore, anintermediate step is inserted between the first and second IHCreactions to elute the primary antibodies of the first reactionwith potassium permanganate or a solution of glycine-HCl forseveral hours or HIER.89,384,387 The use of glycine-HCl withSDS at pH 2 as an elution agent for sequential dual labeling didnot reduce immunoreactivity.274 The elution step could beomitted by developing the first reaction with a concentrateddiaminobenzidine solution, which theoretically would blockany residual primary antibody of the first immunoreaction.89

There are commercial kits with an elution or HIER stepbetween the first and second immunologic reactions.382

Sequential double-labeling techniques are not recommendedif mixed-colored products as a result of colocalization of anti-gens are expected.382 In other words, sequential dual labeling ismore appropriate for the detection of antigens in 2 different cellpopulations or cell compartments (eg, nucleus and cytoplasmicmembrane). A novel IHC procedure can simultaneouslyvisualize 5 biomarkers within a single tissue section using thesame chromogen (amino-9-ethylcarbazole [AEC]) and anti-body elution with KMnO4 and H2SO4.122

Multiple immunolabeling requires stringent controls andcareful combination of enzymes and chromogens to achieve thebest color discrimination (contrast) of the IHC reaction. Theoptimal order of antigen detection depends on several factors,including the need for AR. The choice of counterstain dependsmainly on the color of the immune reaction; it should lightlystain the tissues and differ sufficiently in color from that of thechromogen precipitate to avoid confusion. The most frequentlyused counterstains are hematoxylin, methyl green, and nuclearfast red.390 In some situations, particularly with nuclear anti-gens in small amounts, counterstaining is not recommended.284

The Color of the Antigen-Antibody Reaction. The antigen-antibodyreaction is not visible under the microscope unless labeled. Themost common labels are enzymes, particularly peroxidase andalkaline phosphatase. Each enzyme has specific substrates andchromogens to produce a colored precipitate. The commonchromogens impart a brown, red, or blue color to the reaction.The choice of enzyme and chromogen depends on severalfactors, such as the reaction intensity, antigen location, pres-ence or absence of endogenous pigments, or mounting mediaused, but often is a matter of personal preference.390 Manylaboratories prefer peroxidase, but alkaline phosphatase (AP)is also commonly used and is claimed to be more sensitive thansimilar immunoperoxidase methods.229,230 For horseradish per-oxidase, 3,30-diaminobenzidine tetrachloride (DAB) is theusual chromogen, imparting a brown color that is insoluble inorganic solvents.284 When endogenous peroxidase activity ishigh or melanin pigment is prominent, other chromogens oralkaline phosphatase should be used.370 3-AEC, anotherchromogen for peroxidase, produces a red color. However,coverslips must be mounted with a water-soluble mediumbecause AEC precipitate will wash out with organic solvents.

4-Chloro-1-naphthol forms a blue precipitate that is alsosoluble in organic solvents. For alkaline phosphatase IHC,5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazoliumchloride (BCIP/NBT) (blue, permanent media), fast red (red,aqueous mounting media), and new fuchsin (fuchsia, perma-nent media) are the usual chromogens.284 Alkaline phosphataseis recommended for immunocytochemistry.31 Van der Loos385

reviewed the chromogens used in multiplex IHC with photonicmicroscope and spectral analysis. Newer chromogens are morestable and produce a wider range of colors of the final product.

Multispectral Analysis

Selecting the right set of substrates for multiplex IHC can bedifficult, particularly when antigen colocalization causesmixing of the colored reaction. A novel alternative approachto photonic multiplex IHC is multispectral analysis, in whichthe colors produced during IHC are converted to a spectrumanalyzed by computer.105,384 Multispectral analysis is alsosemiquantitative.384

Quantum dots (QDs) are semiconductor nanoparticles thatcan be used in multiplex IHC. QDs emit different wavelengthsdepending on their size (1–10 nm), shape, and composi-tion.174,268,270,430 QDs can be linked to antibodies, oligonucleo-tides, aptamers, and streptavidin for specific binding to tissuebiomarkers430 and are particularly useful to study tumorheterogeneity.174,214,215 Advantages of QDs in comparison tostandard organic fluorochromes include narrow emission spec-tra (less interference in multiplex analysis), higher photostabil-ity (at least 10 hours), and higher fluorescent signal per unit oflight absorbed.98,268,430 In addition, QD IHC is more accurateand precise at low protein concentration than traditional IHC.61

Refined techniques combining multispectral analysis andtissue microarrays have made high-throughput biomarkerquantification and colocalization more accurate and less cum-bersome.102,163,226 These techniques are described in moredetail in another article.219

Manual vs Automated Immunohistochemistry

Laboratories performing IHC on a routine basis benefit fromautomated systems. Automated IHC follows the same proce-dure as manual IHC, but the procedure is programmed into acomputer that controls the steps executed by the autostainer.Automated IHC saves technician time and typically producesmore consistent results. It has evolved from rudimentarymachines to refined models that can perform the entire proce-dure, even multiplex IHC, without human intervention.

Selection of the best autostainer depends on the laboratory’sneeds in terms of the number of IHC tests, procedural complex-ity, and whether the same platform is to be used for other typesof testing (eg, in situ hybridization). Once the needs for IHC aredefined, the choice of platform may be reduced to a few com-mercially available instruments. This review does not addressthe advantages/disadvantages of each automated IHC platform




but focuses instead on the factors to consider before purchasingan autostainer.

The number of slides that can fit in an autostainer is critical.Small units accommodate 20 to 30 slides at a time. Additionalunits can be connected and controlled by the same computer,providing flexibility if demand for testing increases. ‘‘Closed’’systems versus ‘‘open’’ systems is one of the most hotly debatedchoices in IHC platforms. With a closed IHC platform, mostreagents and consumables (pipette tips, slide holders, etc) mustbe purchased from the platform’s manufacturer. With an opensystem, reagents (including glass slides) from other companiescan be used without affecting the quality of the IHC reaction. Inreality, some platforms classified as ‘‘open’’ are only a bit moreopen than a closed one. Closed systems are favored by labora-tories that run numerous IHC tests on human tissues with littletime for tweaking procedures. It is precisely because most IHCreagents have been developed for use in human tissues that theclosed system has limited appeal in veterinary medicine, exceptfor certain regulatory tests (eg, prion testing), in which numer-ous samples are run simultaneously using a procedure validatedfor a particular IHC platform. For routine veterinary IHC, anopen system allows the necessary flexibility to perform IHCin a wide variety of animal species with both commercial andhomemade reagents.

‘‘Online’’ vs ‘‘Offline’’ Antigen Retrieval. Some platforms allow HIERas a part of the IHC procedure.251 Although this feature providesgreater flexibility, at least with standard AR, these platforms aremore expensive and require higher maintenance that those with-out ‘‘online’’ HIER. Several platforms are designed for continu-ous access, meaning that, as some tests are finalized and theslides are removed from the stainer, a new batch of slides canbe added without stopping the IHC run. This feature is valuedin human clinical laboratories with high demand for rapid test-ing. Other platforms, especially those with ISH capability, canuse higher incubation temperature (eg, 37!C) to increase reac-tion speed. Reagent volume may determine the cost of a test;some platforms require about 100 ml reagent per slide regardlessof the surface area to be covered, whereas others need up to 3times more reagent. The need for special devices to allowincubations with smaller reagent volumes should be consideredin the final cost.

Perhaps the most important factor in selecting an autostaineris the company behind it—in other words, the quality oftechnical service and responsiveness of the company to thelaboratory’s needs. The most sophisticated machine is of littlevalue if the service provided by the manufacturer is suboptimal.It is uncommon, particularly in veterinary medicine, for themanufacturer’s software package to satisfy all the needs of aparticular laboratory. The willingness of a manufacturer to cus-tomize the software to the individual laboratory’s needs isimportant in purchasing decisions. Calculation of the cost perslide produced with an autostainer must include the annual/semiannual maintenance service fee. As Rodney Miller,‘‘Although automated immunostainers have improved the qual-ity of immunostains in many laboratories, the machines are not

perfect, and it is naıve to assume that obtaining an automatedinstrument will guarantee quality and reproducibility.’’239

Storage and Handling of Reagents

The shelf-life of IHC reagents varies depending on their natureand storage conditions. Many primary antibodies and detectionkits can be used beyond the manufacturer’s expiration datewith proper handling and storage.11,323,377 However, such usemust be properly documented.

Diluent Buffer

Antibodies are attracted to the epitopes of most antigensinitially through electrostatic charges and subsequently throughvan der Waals and hydrophobic interactions.32 The isoelectricpoint (pI) of polyclonal IgG ranges from 6.0 to 9.5, so theantigen-antibody reaction is relatively stable at pH 6.5 to 8.5when using whole sera as primary antibody. Although IgGMAbs have a similar pI range,143 slight fluctuations in pH canadversely affect antigen binding.2,427 Reportedly,408 MAbsperform better in phosphate-buffered saline (PBS) than in Trisbuffer. This is puzzling because sodium ions (responsible forthe ionic strength of a buffer) tend to shield negatively chargedepitopes, thereby diminishing the attraction to positivelycharged paratopes of the antibody, especially with alkaline buf-fers.143 Phosphate ions, on the other hand, promote hydropho-bic binding, which could explain the superiority of PBS insome instances. Many authors conclude that the best diluentbuffer for MAbs and PAbs is 0.05 to 0.1 M Tris buffer (pH6.0), but there are exceptions.31,33,429 Tris buffer should alsobe used for alkaline phosphatase procedures, because the highconcentration of inorganic phosphate competes for the phos-phatase.89 Background reactivity due to ionic interactions canbe reduced by increasing the NaCl concentration in the bufferto 0.3 to 0.5 M;277 however, increased ionic strength candisrupt the binding of specific but low-affinity antibodies, soit should be used judiciously.

Assay Standardization

Standardization determines the optimal conditions (eg, incuba-tion time, incubation temperature, dilutions, pretreatment tech-niques, controls, buffers, detection system) for an IHCprotocol. In IHC, the goal of standardization is reproducibleand consistent results within each laboratory and comparableresults among laboratories even with different procedures.291

Unfortunately, with the exception of the surveillance programfor prion diseases, neither the protocols nor the primary antibo-dies are uniform among diagnostic laboratories. This lack ofstandardization of IHC tests among veterinary laboratories284

is complicated by the scarcity of high-quality antibodiesdesigned for the infectious agents and cell markers ofanimals.291 Particularly in tumor diagnostics, most veterinarylaboratories use batteries of antibodies developed for use inhuman tissue.




Even in human medicine, there is little guidance or regula-tion for standardization of IHC tests, although a consensus onguidelines for quality assurance for IHC has been publishedby the CLSI, formerly the National Committee on ClinicalLaboratory Standards.149 Based on the assumption that mostIHC tests are part of the pathologist’s report and not reportedseparately,291 the Food and Drug Administration (FDA) hasclassified most IHC reagents and kits as ‘‘Analyte SpecificReagents,’’ thereby exempting them from premarket notifica-tion. For antibodies used as ‘‘stand-alone tests’’ in humanpathology, premarket notification, and specific FDA approvalare required. For some stand-alone tests in veterinary IHC(e.g., detection of persistent bovine viral diarrhea [BVD] virusinfections), premarket notification by regulatory agencies (USDepartment of Agriculture [USDA]) is not required. However,for the transmissible spongiform encephalopathies, in whichIHC is a stand-alone test for the prion protein, the USDAmandates a standardized procedure, depending on the IHC plat-form, among laboratories.

Practical Standardization of a New Antibody. Tissue preservationaffects performance of the primary antibody. Frozen sectionstypically require less incubation time than FFPE sections and,depending on the fixative, may not require AR. Althoughseveral commercially available fixatives reportedly preserveepitopes for IHC better than formalin, they are more expensiveand often less suitable for routine microscopy.291

Primary antibody characterization. The use of a primary anti-body in a species other than the intended one requires scrutinyof the specification sheet and additional testing. A Western blotcan be used to demonstrate reactivity with the appropriatemolecular weight antigen in a particular species; however,Western blot immunoreactivity does not necessarily predictimmunoreactivity in FFPE tissues. Comparison of immunor-eactivity with that in the ‘‘gold standard’’ species is necessary,but antigen distribution can vary among species. For viruses,immunoreactivity should be detected in the appropriate tissues,cells, and cellular location, as well as in association withtypical lesions. For protozoa and bacteria, consistent morphol-ogy and location of the labeled organisms supports IHC results.For cell markers, reactivity should be detected in the appropri-ate cell or tissue compartment (Fig. 19). More information onprimary antibody evaluation is in the section on test validation.

Antibody dilution. The optimal dilution of the primary anti-body is that titer with the highest ratio of ‘‘signal’’ (specificreaction) to ‘‘noise’’ (background labeling) (Fig. 20).149 Theoptimal titer should produce appreciable differences in labelingintensity within and among samples such that strong- andweak-positive reactions are distinct from the negativereaction.149 This dynamic range of labeling is particularlyimportant in scoring immunoreactivity for prognostic or thera-peutic decisions.149

Primary antibody dilutions for one type of tissue may not beoptimal for another type. The ionic strength of the diluent canbe adjusted to improve the signal-to-noise ratio, particularly for

PAbs in liver sections.431 In a diagnostic setting, however, theoptimal working titer should accommodate a range of relevanttissues. Tissue processing for IHC must be standardized tomatch that used in standardization tests to avoid effects on theoptimal titer.

The manufacturer’s data on antibody performance in FFPEtissue can be used as a guideline for the starting dilution.Depending on the antibody type (monoclonal or polyclonal)and quality, the working concentration should be 1 to 5 mg/mland can be reduced with AR procedures.47 Secondary antibo-dies (working concentration, 5–10 mg/ml47) and detection com-plexes in commercial kits do not need to be titrated.291

To determine optimal dilution of a new antibody, the fol-lowing 3 sets of 5 slides each are recommended: 1 set withoutAR, 1 set with enzymatic AR, and 1 set with HIER. The first 4slides in each set have sequential 2-fold dilutions of the primaryantibody (typically, the midrange dilution is that recommendedby the manufacturer); the fifth slide is incubated with the neg-ative reagent control (Fig. 21).54,287,291,303 A similar approachis recommended by the College of American Pathologists.157

Incubation conditions. The conditions for incubation of theprimary antibody depend on its affinity, ambient temperature,section characteristics, and procedure.291 Incubation for mostroutine protocols is 30 to 90 minutes at RT. Incubating sectionsmust be completely covered by an adequate volume of the anti-body solution to ensure even exposure of the tissue and preventdrying. Shorter incubation at 37!C to 40!C, longer incubationat RT, or extended (overnight) incubation at 4!C can optimizeimmunolabeling.37 Slides should be incubated in a chamber toprevent evaporation of the antibody solution.149 For longerincubations and to reduce evaporation, a container of bufferor water can be placed within the incubation chamber.149

Many antibody traits affect incubation time. Specificity isimportant in selecting the best duration of incubation. Loweraffinity antibodies require longer incubation times, higher con-centrations, or higher incubation temperature.291 Different lotsof the same antibody may vary in concentration or other char-acteristics and thus require re-titration.

Interspecies variations in IHC reactions result from subtlechanges in the amino acid sequence of the targeted antigen(Fig. 22).290 Even with proven interspecies cross-reactivity, theantibody affinity may be decreased in a different species. Pro-longed incubation, adjusted AR, and/or increased antibodyconcentration may be needed for optimal IHC. Antibody speci-ficity must be verified in each species tested.291

Choice of detection system. Many commercial detection kits,some optimized for particular animal species, claim to be themost sensitive, but any detection kit must be validated in housebefore routine use. The secondary and tertiary reagents ofthese kits may contain antibodies or other compounds thatcould react nonspecifically with tissue antigens, increasingbackground or producing false-positive results. This is one jus-tification for negative controls in IHC (see below in validation).Typically, a non–biotin-labeled polymer detection system is




Figure 19. Each antigen has a typical anatomic compartment. (a) Carcinoma, mouth; dog. Cytoplasmic pattern for cytokeratins. (b) Lymphoma,lymph node; dog. Cell membrane location of CD20. (c) Histiocytic sarcoma, spleen; dog. Although CD18 is a cell membrane antigen, it may beexpressed in a paranuclear (Golgi) location. Inset: Detail of the reaction. (d) Plasmacytoma, skin; dog. The transcription factor MUM1 isexpressed in plasma cell nuclei. Inset: Detail of reaction. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 20. Optimal titrationof the primary antibody (Ab). Lymph node; dog. Prox-1 is a transcription factor of lymphatic endothelial cells. (a) Excess Ab (1/100 dilution)




recommended if HIER is used to avoid background fromEABA.54

Postanalytical Phase of IHC

Assay Validation

Validation is a process to affirm the suitability of an assay, onceit has been developed, optimized, and standardized, for a spe-cific purpose.419 Validation appraises the analytical and diag-nostic performance of a test.419 A validated IHC test shouldbe reproducible with high sensitivity and specificity.71 Valida-tion entails several steps starting with evaluation of analyticalcharacteristics: specificity and sensitivity, repeatability, andpreliminary reproducibility. 419 Next, diagnostic characteris-tics, particularly diagnostic specificity and sensitivity, and cut-off value are evaluated. Finally, the test’s reproducibility andruggedness are evaluated with implementation among labora-tories and continuous monitoring of validation criteria.419 Testvalidation requires proof of antibody specificity in an estab-lished assay. For most IHC tests, this involves detecting anycross-reactivity of the antibody with unrelated antigens (or thepresence of antigen in a cell or tissue not previously known toharbor it) or among different tissues species. Moreover, valida-tion examines the variables that affect the IHC reaction, such asfixation time and storage of paraffin blocks or unused tissuesections.291 A single assay may be validated for several pur-poses by optimizing for each intended purpose.419 For exam-ple, if an IHC marker (analyte) is to be used to demonstratethe same antigen in different animal species, antibody dilution,incubation time, AR, and other test parameters may needadjustment. An IHC test can be validated by comparing resultsamong laboratories using similar techniques.16 When possible,validation compares the IHC test sensitivity to that of the ‘‘goldstandard’’ method for the antigen in question. It may also com-pare staining patterns and sensitivity with other antibodies tar-geting the same antigen. Test validation follows teststandardization.419 The CLSI evaluation protocol for a qualita-tive diagnostic test, such as IHC, in situ hybridization andWestern blotting analysis of 50 positive and 50 negative speci-mens if validation data are not available from other sources (eg,antibody manufacturer).71 This target can be difficult toachieve in veterinary medicine where validation may have tobe an ongoing process. The in-house validation proceduredepends on the antibody and the laboratory. A validated IHC

assay should produce consistent results, regardless of themethod or the laboratory where it is performed.291

As mentioned earlier, validation of an IHC test may requirean additional method (eg, the gold standard) to prove the pres-ence or absence of the antigen in question. In some cases, thegold standard method does not prove the presence of a proteinbut demonstrates a physiological activity closely associatedwith the given protein. An example is the validation by Marti-net et al228 of an assay to demonstrate autophagy-related pro-teins. The gold standard to detect autophagy is transmissionelectron microscopy (TEM). However, because this methodis time-consuming and not routine, the authors developed anIHC assay. Autophagy was induced in mice by starvation (pos-itive control, confirmed by TEM); the negative control was tis-sue from transgenic mice with the essential autophagy geneAtg7 deleted. These mice lacked autophagolysosomes by TEM,even when starved. Different commercial primary antibodies,putatively specific for autophagy, were used as well as differentfixation procedures, buffers, and AR protocols. The use of Atg7knockout mice as a negative control proved that some of thetested antibodies indeed recognized a different protein innormal and starved mice.

The use of acetone- or ethanol-fixed frozen tissue sectionshas been advocated as the gold standard against which IHCon FFPE should be compared. However, in some instances, fro-zen sections fixed in coagulating fixatives are suboptimal forIHC compared with FFPE tissue sections.338 This is particu-larly relevant when testing low-molecular-weight proteins andlipoproteins, which are readily extracted by coagulating fixa-tives.203 The same conclusion was reached by van der Loos383

in studying the distribution of interferon (IFN) g–producingcells using different fixatives (Fig. 6). This point is corrobo-rated by international governing organizations of diagnosticassays, which indicate that the matrix (eg, tissue section) invalidation studies should be identical or closely resemble thesamples to be tested.419

Primary Antibody Characterization. Validation of an assayrequires confirmation that the primary antibody is specific,selective, and reproducible for its intended use.40 For cellularmarkers, a good starting point is Western blot (WB) to demon-strate reactivity with the appropriate molecular weight antigenin the tissue and species of interest;320,386 multiple bands orbands at inappropriate molecular weight could indicate post-translational modifications or low antibody specificity.40,200,321

results Figure 20. (continued) in muddy nuclear labeling in both endothelial cells and lymphocytes with background in plasma (v). (b) Specificnuclear labeling of Prox-1 in lymphatic endothelium at 1/400 Ab titer. (c) Labeling of lymphatic endothelial cells (arrowheads) with lack of back-ground in lymphocytes or plasma (v) at 1/800 titer. Note hemosiderin in macrophages at bottom (asterisk). Immunoperoxidase-DAB, hema-toxylin counterstain. Figure 21. Antibody standardization for immunohistochemistry (IHC). Three sets of sections (5 slides per set) areused. Two-fold dilutions (1/25 through 1/200) were made (first 4 slides on each row; the last slide is the negative reagent control). Sectionsin each set were treated with no antigen retrieval (AR) (a), enzyme digestion (proteinase K [PK]) (b), or heat-induced epitope retrieval (HIER)with citrate buffer, pH 6.0 (c). Immunoperoxidase-DAB, hematoxylin counterstain. Figure 22. Oral mucosa; horse. IHC with monoclonal anti-bodies (MAbs) to vimentin. (a) Mouse MAb reacted appropriately with mesenchymal cells such as lymphocytes (arrowheads), smooth musclecells of vessels (asterisk), and interstitial cells (solid circle) and did not label epithelial cells. (b) Rabbit MAb had no reactivity. Immunoperoxidase-DAB, hematoxylin counterstain.




Nevertheless, WB immunoreactivity does not necessarilypredict immunoreactivity in FFPE tissues; WB requires antigendenaturation (therefore, modification of its secondary and ter-tiary structure) as opposed to the in situ antigen modificationin formalin-fixed tissues, with cross-linking rather than dena-turation.55,413 Some antibodies bind only denatured proteinimmunoblots; others bind only native proteins.55,69 In somecases, as with HIER, the antigen denaturation by WB may notbe problematic and does not disqualify this procedure, becauseIHC detection of most epitopes requires only an intact primary(linear) protein structure.39,347

Incubating the antibody with excessive blocking peptidesbefore the IHC reaction (the so-called preadsorption test) hasbeen used in validation.40,155 However, blocking peptide proce-dures do not prove antibody specificity or provide informationregarding signal-to-noise ratios,360 and the amount of antigenthat constitutes an ‘‘excess’’ can only be calculated by knowingthe Kd of the antibody.321 Diluting the antibody to the point thatlabeling occurs only in expected locations is a more logicalapproach.321

Despite their shortcomings, WB and preadsorption tests arethe most widely used methods to determine the specificity of aprimary antibody in a diagnostic setting. Other methods thatrequire more sophisticated technology and laboratory capabil-ities include (1) use of knockout animal tissues to demonstratethe lack of detection of the protein genetically removed fromthat animal and (2) use of a transfected cell line expressing theantigen for the primary antibody.55,320 Comparison of immu-noreactivity with the ‘‘gold standard’’ species is another option;however, antigen distribution may vary among species.291 Inthe end, antibody specificity is best documented by the appro-priate use of controls (see below).40

A special situation arises with commercial purified affinityMAbs that claim specific antigen targeting as demonstrated byWB on a limited set of tissues or cell culture extracts. By def-inition, these antibodies are specific for a particular epitope. Inreality, some of these ‘‘commercially validated’’ antibodiescross-react with unrelated antigens associated with degeneracyand immune mimicry.63,279 This unexpected cross-reactivity isdifficult to detect without extensive antibody validation withdifferent tissues by WB or paraffin sections.386 For mostlaboratories, this approach is neither practical nor economicallyfeasible.

A common practice in the biopharmaceutical industry is tis-sue cross-reactivity (TCR) studies to identify off-target bindingof primary antibodies or antibody-like molecules with acomplementary-determining region; TCR is also used to detectpreviously unidentified sites of on-target binding.204 Thesepharmaceutical compounds, intended as vehicles of biologi-cally active molecules, are incubated with microarrays ofhuman or animal tissues (frozen or, less commonly, fixed);TCR studies supplement but do not replace in vivo studies.204

In some laboratories, the same MAb (same clone andantibody presentation) from 2 different companies is used inthe same IHC procedure for validation purposes. We haveexperienced aberrant immunoreactivity using an antibody to

CK8/18 from one company, whereas the same clone from a dif-ferent company produced the expected results (Figs. 23, 24).This discrepancy suggests that nonactive ingredients of themonoclonal preparation could have interfered with the immu-nologic reaction.

Cross-Reactivity. Cross-reaction can result in a false-positivereaction.419 However, in IHC, 2 main types of cross-reaction,neither of which necessarily implies a false-positive reaction,can be used to the diagnostician’s advantage within the propercontext: antigen cross-reactivity and species cross-reactivity. Itis assumed that test conditions are those used duringstandardization.291

Antigen cross-reactivity. The structure and amino acidsequence of an antigen affects its cross-reactivity. A literaturesearch may reveal reports of antigen cross-reactivity. Lack ofcross-reactivity in one species does not preclude it inanother.291 Molecular mimicry (eg, cross-reactions of infec-tious agent antigens with normal tissue antigens) may alsooccur.353

Evaluation of cross-reactivity in the diagnostic settingdepends on the category of the antibody (against infectiousagents vs cell markers).291 Antibodies against bacteria, proto-zoa, or fungi should be tested against other microorganismsthat are morphologically similar, belong to the same group,or produce similar lesions in the organ of interest. Antibodiesto viruses should be tested against other viruses in the samegroup that affect the same tissue or produce similar lesions.Some lack in specificity does not preclude the use of anantibody, provided it is documented and interpreted. For exam-ple, an antiserum against porcine coronavirus is used to detectfeline enteric coronavirus, feline infectious peritonitis virus, ormink and ferret coronaviruses. Some MAbs against infectiousagents cross-react with cellular organelles.

For neoplasms, antibodies to specific cell types or compo-nents should be evaluated in 1 or more of the following ways:(1) comparison of immunoreactivity in primary versus second-ary (metastatic) tumors (Fig. 25),288,297 (2) determination ofimmunoreactivity in other neoplasms in the differential diagno-sis,289,294,296,297,299,302 (3) comparison of immunoreactivitybetween nonneoplastic and neoplastic tissues (antigen expres-sion may be lost or expressed de novo in neoplastic cells)301

or between benign and malignant phenotypes,392 (4) evaluationof immunoreactivity in tumors with different histologicpatterns or cellular phenotypes,288,295,297 (5) determination ofantibody cross-reactivity between neoplastic and nonneoplasticcells in the organ of interest (eg, hepatocellular tumors vs sec-ondary hepatic tumors),298 (6) comparison of reactivitybetween wild-type and mutated antigens (eg, p53), and(7) semiquantitative evaluation of immunoreactivity of variousantibodies targeting the same cell or cell product in varioustumors or tissues to determine their relative diagnostic utility(Fig. 26).290,301,302

Species cross-reactivity. Although some antigens can bedetected under similar conditions in different species, it should




Figure 23. Manufacturer differences in MAbs. Intestine; dog. MAb 5D3 cell culture supernatant for cytokeratins (CK) 8/18. Compare with Fig.24, from a different manufacturer. (a) Expected reactivity in mucosal epithelial cells (solid circle) with no labeling of mesenchymal cells in thesubmucosa (s) or muscularis (m). (b) Detail of the mucosa. (c) Detail of the submucosa and muscularis. Immunoperoxidase-DAB, hematoxylincounterstain. Figure 24. Manufacturer differences in MAbs. Intestine; dog. MAb 5D3 cell culture supernatant for CK 8/18. Compare with Fig.23, from a different manufacturer. (a) Intense labeling of mucosal epithelial cells as well as many mesenchymal cells, including lamina proprialleukocytes (solid circle), fibroblasts, vessel walls, and nerves in submucosa (s) and muscularis (m). (b) Detail of lamina propria immunoreactivity.(c) Detail of submucosal immunoreactivity. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 25. Antigen cross-reactivity. Intestine;dog. The B-lymphocyte marker CD79a is strongly expressed in normal smooth muscle of the submucosa (s) and muscularis (m).Immunoperoxidase-DAB, hematoxylin counterstain. Figure 26. Antibody interspecies cross-reactivity. Skin; horse. (a) Melanocyte markerPNL2 labels neoplastic cells of equine melanoma. (b) Melanocyte marker Melan A is not expressed in equine melanoma. Insets: Immunohisto-chemistry detail. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 27. Infectious bovine rhinotracheitis virus (IBR) infection, colon;calf. Necrotic foci in lamina propia (l) and submucosa (s and arrowheads) have strong IBR antigen expression. Inset: Epithelial cells havenonspecific supranuclear expression in the Golgi zone. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 28. Inadequate heat-induced epitope retrieval (HIER). Liver; horse. (a) Leakage of solution during antigen retrieval left the upper third of the tissue section unex-posed to HIER solution, therefore unable to react with the primary Ab. (b, c) Reactivity was minimal in the portion of the section not exposed toHIER solution (solid circle). Immunoperoxidase-DAB for equine herpesvirus 1, hematoxylin counterstain.




be assumed that antigen detection varies among species.301

Identical antibody clones differ in reactivity among species.Not all antibodies targeting particular antigens will be reactivein all species. For example, Melan A labels melanocytes indogs, cats, and other species but not in horses.290 It is advisableto restandardize (optimize) an IHC test for each new species,including incubation times, concentration of the primaryantibody, and AR. A literature search or consultation with theantibody manufacturer and extensive testing of similar cases ina given species are recommended to determine species cross-reactivity.291 A diagnostic IHC report should indicate thedegree of confidence in the results based on experience andpublications. The use of tissue arrays from different animalspecies permits rapid screening for interspecies cross-reactivity.173,265,284

Effects of Fixation, Postfixation Treatments, and Equipment onImmunoreactivity. These laboratory variables affect assay devel-opment and validation.419 Different fixatives have differenteffects on immunoreactivity. For each fixative, full standardi-zation of a test must be done, including incubation conditions,dilution of the primary antibody, and AR method (see sectionon standardization). Standardization is usually done on tissueswith known fixation time (eg, 1–2 days).

Fixation kinetics studies are recommended for each antigenor epitope as part of the validation process to determine theassay robustness, defined as its capacity to produce reproduci-ble results despite small variations in method parameters (eg,incubation temperature, pH of buffers, batch of reagents, shelflives and storage conditions, fixation length).419 A positivetissue control should be fixed for different durations (eg, 1, 2,4, 7, 10, 14, 21, or 20 days) and tested under identical condi-tions to determine the effects of fixation time on reactivity(ie, intensity and number of cells or organisms detected(Fig. 3).288,297,403,404

Automated stainers are designed to duplicate manualprocedures and ensure uniform application of each step of theprocess. Thus, the use of automated equipment creates a uniformand standardized testing environment, which will improve intra-laboratory run-to-run consistency.251,369,433 When results amongdifferent laboratories diverge considerably, technical differencesshould be considered as a possible cause.

What Constitutes a Positive/Significant Result? There is no singleanswer to this question. IHC assays detect analytes (infectiousagents or tumor marker antigens) in tissues, but diagnosisrequires interpretation of IHC results in the context of clinical,pathologic, or laboratory findings (the so-called class I IHCtests).375 For an infectious agent, detection of even 1 organismindicates infection, but it is another matter to prove that theagent caused disease. For neoplasms, interpretation is evenmore complex, because tumors are often heterogeneous, andtumor stem cells can differentiate in various directions. Thereported percentage of positive cells required to confirm tissueorigin of a tumor varies considerably. A cutoff value of 10%positive cells has been suggested to classify an IHC test as

positive,375 but the cutoff value is by no means set in stone.136

Some pathologists prefer the words detected or not detectedrather than positive or negative, which underscores the need forsubjective interpretation of the IHC reaction in the context ofthe disease.326 Each antigen has a ‘‘different weight’’ in thefinal interpretation. A more pragmatic approach would be to seta cutoff value for each antibody, optimized for a given entity.For example, the cutoff value for CD34, consistently expressedin many different mesenchymal cells, should be higher thanthat for uroplakin III, which, although highly specific forurothelial tumors, may be expressed in only a small proportionof the neoplastic cells.297 For prognostic markers or those usedto customize therapy, a standardized scoring system applicableto different platforms is required for meaningful results.380,402

How to Distinguish True-Positive From False-Positive Immunoreactiv-ity. The pattern of immunoreactivity, particularly with neo-plasms, is important in distinguishing a true-positive reaction,which has some degree of cell-to-cell heterogeneity, from afalse-positive reaction, which lacks heterogeneity, even in theabsence of stromal background reactivity.239 Reactivity of abiomarker in tissues/cells not reported previously is notunusual; less commonly, an antigen is detected in a location notconsistent with its function. For example, thyroid transcriptionfactor 1 (TTF1) expression is expected in the nucleus, but notcytoplasm, of thyroid and pulmonary neoplasms;299,300 how-ever, cytoplasmic TTF1 expression has been reported in humanliver neoplasms and confirmed in mitochondria by other meth-ods.58,267,410 Whether the detected antigen is part of the TTF1molecule or a cross-reacting antigen is not clear.

Clinical Validation. Clinical validation is based on whether a testresult correlates with case outcome or response to therapy.126

Clinical validation is common in human oncology but requiresaccess to hospital medical records from numerous cases. Inveterinary medicine, prognostic or therapeutic assumptions areoften based on published information.24,191,319,373,395

Controls in Immunohistochemistry

Positive Tissue Control. A positive tissue control is a tissue knownto contain the targeted antigen detectable by identical IHCmethods to those used in diagnostic cases.149 Positive tissuecontrols assess the performance of the primary antibody.149

Fresh-fixed surgical biopsy specimens are preferred overpostmortem specimens, because extended warm ischemia andautolysis affect immunoreactivity.149 For laboratory animals,autolysis is less of an issue, and postmortem specimens are rou-tinely used. Positive tissue controls must be fixed, processed,and stained in the same way as the test tissue for each antibodyand procedure.306,369 Control tissues should be from the samespecies as the test tissue, with few exceptions (see below). Theantigen in the positive control should not be abundant andshould have some heterogeneity in intensity throughout thesample; weakly immunoreactive areas can be used to detectsubtle changes in antibody sensitivity.369 A positive control




with abundant antigen may reduce the chances of identifyingweak-positive cases.149 The presence of the antigen in thetissue control should be confirmed by another method (eg,PCR, virus isolation) or documented by publication. For mostlaboratories, control tissues are generally obtained ‘‘in house’’because fixation and processing procedures are those used withthe test tissue. Control tissues from another laboratory or acommercial source could have been fixed or processed differ-ently. When possible, the positive tissue control should be onthe same slide as the test tissue to minimize technical variation.

Multitissue blocks and cell lines. Various methods for thesimultaneous study of multiple FFPE tissues in a single histo-logic section have been reported. In the sausage technique,17

long thin strips of fixed tissues are drawn into a tube of unfixedsmall intestine. The entire sample is then fixed, resulting in atight column that can be cut into blocks and embedded.Substitution of placenta as the ‘‘wrapping’’ tissue115 and othermodifications239 to the original method have been reported.

Paraffin-embedded tissue microarrays. These have been used ascontrols in human IHC laboratories, mainly for tumor diagno-sis.158,265,266,284 Validation studies should be carried out onmultitissue control blocks containing both known-positive andknown-negative normal and neoplastic tissues.369 In veterinarymedicine, species-specific tissue microarrays are not commer-cially available. Species-specific tumor cell lines can be usedfor IHC controls (eg, for HER-2/neu);306,310 this approachprovides an identical control for comparison of results amonglaboratories.291

Peptide controls. The basis behind peptide controls is thatformalin-fixed peptide epitopes, covalently attached to glassslides, behave immunochemically like the native protein in tissuesections.38 Mass production of peptide controls is cheaper thantissue or cell line controls, creates an identical control for numer-ous assays, and facilitates standardization of IHC protocols.

Reference control tissues for infectious diseases. The presence orabsence of an infectious agent in a tissue should be assessed byknown IHC reactivity patterns plus another (non-IHC) diagnosticmethod.15 For example, Listeria monocytogenes control tissuecould be assessed by IHC (immunoreactivity of extracellular orphagocytized bacilli) and by bacterial culture. Similarly, BVDvirus control tissue could be assessed by IHC (immunoreactivityin cytoplasm of infected cells) and by virus isolation or PCR. Inaddition, the presence of characteristic lesions of a particular dis-ease supports use as a standard reference control.291

IHC protocols for infectious diseases should be speciesmatched with test specimens; alternatively, tissues from otherspecies infected with the same agent can be used (eg, BVD virusidentification in test tissue from cervids using a bovine control tis-sue). Nonspecific binding of primary antibody (species-specificmolecular mimicry) or secondary (linker) reagents can occuramong species. An example is the use of goat PAbs against Neos-pora caninum on goat tissue. The anti–goat IgG secondaryreagent can bind extensively to endogenous goat IgG in the tissue,

resulting in background ‘‘noise’’ that obscures any immunoreac-tive tachyzoites.291

Reference controls for neoplastic diseases. Often, the only wayto verify the presence of an antigen is by histology, which is notalways accurate. Moreover, a negative result for tumor markersdoes not rule out a particular neoplasm because the marker ofinterest may not be expressed in a poorly differentiated cellpopulation. Ideally, controls for tumor cell markers shouldconsist of the neoplasm of interest, one with heterogeneousreactivity.369 Control tissues for IHC tumor markers shouldalso be species matched with test specimens because the detec-tion of an antigen in a particular tumor and animal species doesnot guarantee similar results in a different species.291

Negative Tissue Control. A negative tissue control is known notto contain the antigen of interest.149,369 A negative controlshould be included in each IHC run to verify the specificityof the primary antibody and the presence or absence of back-ground (false-positive) reactivity; if false-positive reactivity(tissue labeling with a pattern similar or identical to that in thepositive control) occurs in the negative control, the test shouldbe considered invalid.149 At least one ancillary test (eg, PCR,virus isolation) on the same animal should be used to rule outthe presence of the antigen of interest. As for the positivecontrol, a negative tissue control should be processed in thesame manner as the case material. Similarly, when using multi-tissue blocks for antibody validation studies or as tissue con-trols, the specimens must be fixed and processed as for thecase material.369 In practice, only 1 tissue control is used dueto the common presence of both positive and negative cellswithin the positive control. If possible, the positive tissue con-trol section should be on the same slide bearing the diagnostic(test) tissue section.291

The lack of labeling with a negative tissue control does notnecessarily validate labeling in the positive tissue control. Anexample is observed with some MAbs produced in ascites fluidlabeling the Golgi zone of different types of epithelial cells indifferent species (Fig. 27).351 This nonspecific labeling is morerelated to the immunized mouse rather than to the type ofimmunogen used or the immunoglobulin class (eg, IgG1 orIgG3) produced in the ascites fluid and has been blocked byadsorbing the ascites fluid with blood group A1 human erythro-cytes.351 We have observed similar nonspecific reactivity withsome MAbs. In the end, interpretation of specificity is based onthe expected location of the antigen in a particular cell or cellcompartment.400

Internal Positive Tissue Controls. Internal positive tissue controls(eg, smooth muscle markers or vimentin in blood vessels) arepresent in many test tissues. Labeling in these areas indicatesappropriate immunoreactivity. In addition, with internalcontrols, there is no variation in fixation or processing.32,239

Internal controls can be used to assess sample quality byidentifying ‘‘housekeeping’’ or structural proteins present inrelatively constant amounts in certain cell types in a wide vari-ety of tissues, such as endothelial cells or lymphocytes.369




Reagent (Antibody) Controls. Negative reagent controls (withoutthe primary antibody) are used to confirm test specificity andto assess the degree of nonspecific background caused by thesecondary or tertiary antibody.149 Commonly, the primary anti-body is replaced by 1 of the following: (1) antibody diluent,(2) same-species nonimmune immunoglobulin at the same con-centration and in the same diluent, (3) an irrelevant antibody, or(4) buffer.306,369 Only reagents 2 and 3 qualify as true-negativecontrols.291 Ready-to-use, universal negative reagents are com-mercially available for rabbit and mouse primary antibodies.With the different quality standards among laboratories, anagreement on which reagent control is best may not be forth-coming.400 The current CLIS-approved guideline for IHC alsoaccepts, as an inferior option, simple omission of the primaryantibody from the protocol.149

If the diagnostic workup requires a panel of antibodies, eachof the same isotype and similar concentration and derived fromthe same species, the panel of primary antibodies itself canserve as a set of irrelevant reagent controls; thus, the need formultiple negative controls is eliminated. With this method, sep-arate controls should still be run for each different protocol (eg,different AR or detection system).369

Quality Assurance/Quality Control

Human IHC laboratories must meet federally mandated stan-dards of operation as defined by the Clinical LaboratoryImprovement Amendments (CLIA).101 Veterinary laboratoriesaccredited by the American Association of Veterinary Labora-tory Diagnosticians (AAVLD) must meet the ISO/IEC 17025standards for all types of assays, including IHC. This documentestablishes the general requirements for testing, includinglaboratory organization, document control, test calibration, andtechnical requirements for performing a test. Technical require-ments include personnel training and qualifications, environ-mental conditions, calibration and validation, equipment, andreporting.168 In nonaccredited laboratories, diagnostic testsshould be validated by documentation of internal or interla-boratory performance using reference standards and relevantdiagnostic specimens. This should be corroborated by theendorsement of organizations, such as the World Health Orga-nization, by publication in peer-reviewed journals or by directcomparison with an established method. Following are generalguidelines for quality control (QC)/quality assurance (QA)standards for veterinary laboratories. More detailed informa-tion has been published.291

Daily Quality Assurance/Quality Control. Each laboratory shouldestablish standard operating procedures (SOPs) for histology.These include schedules for cleaning, maintenance, and moni-toring logs, along with a daily check of equipment such asmicrowave, oven temperature for drying slides, temperatureof water baths, pH meter, reagent containers, and so on (Fig.28).217 Buffers, antibody dilutions, and other reagents shouldbe prepared according to standard or manufacturers’ recom-mended procedures.

Internal Quality Assurance/Quality Control. A biannual internal QA/QC check of IHC slide interpretation by pathologists isrecommended.

External Quality Assurance/Quality Control. The goal for interla-boratory comparisons is to document variations in reactivityamong laboratories and to set minimum QA/QC standards forIHC protocols to be shared by all laboratories.409 Despite itsdifficulty, interlaboratory standardization of IHC tests on ani-mal tissues in veterinary laboratories is in progress. Even inhuman medicine, only a handful of IHC tests (eg, Hercep test)have been validated in such a way that different laboratoriescan perform tests with consistent results and not withoutcontroversy.28,156,314 As Fletcher and Fletcher104 pointed out,‘‘Whether we like it or not, the practice of anatomic pathologyis to some extent subjective, although we all strive for as muchobjectivity and reproducibility as possible in our daily work.’’

There are 2 main approaches to interlaboratorystandardization.291

Technical equivalency testing of an IHC test. Unstained slides aredistributed among different laboratories for performance of theIHC test. Technical equivalency testing evaluates the quality andinterlaboratory consistency of IHC and focuses not only on resultsbut also on the technical quality of the IHC preparation and arti-facts that might influence the test results (eg, background staining,loss of tissue due to harsh treatment). Currently, the PathologyCommittee for the AAVLD Program for Interlaboratory Compar-ison of Infectious Agent Immunohistochemical Assays recom-mends an annual, voluntary, immunohistochemical proficiencytest managed by the Pathobiology Section of the National Veter-inary Laboratory Services, USDA (Ames, IA). Unstained paraffinslides are sent to participating laboratories, which perform therequired IHC test and interpret the results. Evaluation includesquality of the IHC procedure and its interpretation. These interla-boratory tests are common in human medicine and have exposeddifferences in IHC proficiency among laboratories that couldaffect therapeutic decisions.68,136,309,380,416,426 For ‘‘stand-alone’’IHC tests in veterinary medicine (eg, scrapie, chronic wasting dis-ease), participating laboratories must pass an annual proficiencytest.291 Similar requirements have been established for variousbiomarkers in human tissues.416,426 External quality assurance forimmunocytochemical preparations is limited.188

Diagnostic equivalency testing using IHC testing as an adjunct. Inthis case, both the pathologist’s proficiency and the technicalquality of IHC are evaluated. A brief history and descriptionof the tissue examined is included with tissue sections. Thepathologist must base the diagnosis on routine (eg, hematoxylinand eosin) staining and support it with appropriate IHC testing.Results could be reviewed by a committee to address anydifferences among participating laboratories.407

Immunohistochemistry Reporting and Interpretation

If not included in the standard report, the IHC report should con-tain demographic information pertinent to the case, the tissue




that was tested, and the antibody used or the targeted antigen.Other IHC test details should be filed in the laboratory. Results forinfectious antigens should be reported as ‘‘detected’’ or ‘‘notdetected.’’ The interpretation for infectious disease IHC/immuno-cytochemistry (ICC) tests should include a disclaimer that anylack of detectable antigen could be due to the absence of theantigen in the section or to technical aspects, such as prolongedfixation or method sensitivity.291 For tumor markers, a descrip-tion should include the cellular location (eg, cytoplasmic, cellmembrane, nuclear) (Table 6), distribution through the tissue sec-tion, labeling intensity, and percent positive cells, along with aninterpretation of the test results.149 If antigen location isatypical, or if testing of a marker has not been validated in the spe-cies of interest, this should be stated in the report.291 A scoringsystem for IHC has been proposed by CLIA (Tables 7, 8).149 Per-sonnel who read IHC slides should be familiar with the antibodiesused and their specificity and sensitivity, and an experiencedpathologist should review daily IHC preparations for QA/QC.291

Most important, interpretation of IHC results requires famil-iarity with the expected pattern of immunoreactivity based onlocation of the antigen in the cell of interest.425 For example,

labeling of cytokeratins and vimentin should be cytoplasmic;labeling of TTF1 in lung or thyroid tumors is nuclear.299,300

The degree of specific immunoreactivity varies among cellsand, in some cases, among different cell compartments. Cellu-lar distribution of some antigens has prognostic significance incertain tumors.191 Finally, interpretation of an IHC reaction isonly as good as the controls.413

Particularly in leukocytic populations, IHC expression of amarker can confirm cell type, without documenting the neo-plastic nature of the proliferation. Molecular demonstrationof clonality supports a diagnosis of neoplasia, for example, infeline intestinal lymphoma, in which histologic distinctionbetween neoplastic lymphocytes and reactive lymphocytes ofinflammatory bowel disease is difficult.185,190,246

Patterns of Specific Labeling. Antigens can be present in variousorganelles and intracellular locations or outside cells (Fig. 19).In the nucleus, viral inclusions, inclusions of known or unknowncause, nucleoli, chromatin, the nucleoplasm, and the nuclearmembrane may be labeled. Other intracellular sites of antigenexpression include mitochondria, lysosomes, rough and smoothendoplasmic reticulum, lipid droplets, peroxisomes, pigment

Table 6. Staining Patterns and Manual Interpretive Scoring Criteria.

Cellular Location Staining Pattern Scoring Criteria

Nuclear only Staining limited to nucleus Positive/negativePercent positiveIntensity

Cytoplasmic only Staining limited to cytoplasm Positive/negativePercent positiveIntensity

Membrane only Staining limited to cell membrane Positive/negativePercentage positive

Nuclear and cytoplasmic Staining in both nucleus and cytoplasm Positive/negativePercent positiveIntensity

Nuclear or cytoplasmic Variable location Cytoplasmic or nuclearPercent in a location

Membrano-cytoplasmic Staining in the cell membrane and the cytoplasm Positive/negativePercent positiveIntensity

Modified and used with permission by the Clinical and Laboratory Standards Institute, document I/LA28-A2.149

Table 7. Immunohistochemical Score Based on Different Percentagesof Cytoplasmic Staining.

Score Interpretation Description

0 0% No staining1 Borderline/background Weak staining in less than 50% of cells2 Positive Strong staining in less than 50% of

cells or weak staining in more than50% of cells

3 Strongly positive Strong staining in more than 50% ofcells

Modified and used with permission by the Clinical and Laboratory StandardsInstitute, document I/LA28-A2.149

Table 8. Scoring Approaches for Immunohistochemistry NuclearStaining.

0–4 Grading Scheme(% Positive Cells)

0–3 Grading Scheme(% Positive Cells)

0 (0) 0 (0)1 (1–25) 1 (1–10)2 (26–50) 2 (11–50)3 (51–75) 3 (51–100)4 (76–100)

Modified and used with permission by the Clinical and Laboratory StandardsInstitute, document I/LA28-A2.149




granules, and other organelles or structures. An antigen may befound in several locations, in several different organelles, in boththe nucleus and cytoplasm, and in the cytoplasmic membraneand cytoplasm. Outside the cell, connective tissue, tissuematrices, extracellular fluids, and other substances both normaland abnormal can be immunoreactive. Antigens can exist in cer-tain anatomical locations in normal cells but in other locations inneoplastic or reactive cells. Genetic changes (eg, mutations) canresult in antigenic shift and redistribution; for instance, b-cateninis typically a cell membrane protein but can be detected in thenucleus in some cases.27,424 Antigens that exist at low levelsor have a short half-life in normal cells (eg, p53) may only bedetectable by IHC after mutation.

Patterns of Nonspecific Labeling. Nonspecific backgroundstaining can mimic specific immunoreactivity. At the highestantibody concentration, all cells (epithelial, mesenchymal, andneural) and tissues are brown (with DAB as chromogen) (Fig.20). With increasing dilution, nonspecific staining disappears.Some cell types, especially mast cells and plasma cells, mayappear brown at all dilutions depending on the IHC technique,but these cells will also be brown in the negative control slides,which were not exposed to the primary antibody. The carefuluse of appropriate controls should allow distinction betweenbackground and specific labeling.

Immunocytochemistry

ICC is the detection of antigens in cytologic preparations bymeans of an immunologic reaction visualized by a chemicalreaction.345 More biomarkers are immunoreactive in cytologicpreparations than in FFPE tissue sections.379 There are impor-tant methodological differences between IHC and ICC in (1) thetype of substrate or sample, (2) storage of unstained samples,(3) fixation, and (4) test validation and controls.86

Type of Cytologic Preparation. ICC can be performed on cytospins,cell smears, cell blocks, cytoscrape cell blocks, cell cultures, andliquid-based monolayer preparations.43,62,86,101,345,435 Cellsmears give reproducible results with nuclear markers but areless suitable for cytoplasmic and membrane markers due to thehigh background produced by cell damage during slide prepara-tion.345 Cytospin preparations are less susceptible than smears tocell damage.284 The use of pretreated slides to promote celladhesion reduces loss of cells during the ICC procedure.86

Liquid-based cytology using ThinPrep preparations enhancesretrieval of cells from small samples with preservation of cellu-lar detail and, theoretically, a reduction of background due toless blood, mucin, and proteinaceous material in the sample.86

Cell blocks are one of the best choices for ICC, particularly whencells are numerous.239,285,286 Cell blocks are processed similarlyto surgical pathology specimens, so IHC methods can be usedwithout modifications.86 In addition, multiple sections can beproduced from a single block, so multiple markers can beevaluated in the same cell population.86,286,394 However, cellblocks can vary in cellular density and detail, depending on the

nature of the lesion, the quality of the fine-needle aspirate, andpostprocedural handling of the needle-rinse specimen.312 Impor-tantly, because cell blocks are fixed in formaldehyde-containingsolutions, sections from cell blocks may need to undergo ARprocedures.45,339 A more detailed discussion of the pros and consof cytologic preparation methods is in Fowler and Lachar.107

ICC can be performed, even repetitively,252 on smearspreviously stained with Romanowsky or Papanicolau and indecolorized smears.1,14,240 However, cell loss, cell disruption(affecting mostly cell membrane and cytoplasmic markers),and antigenic degradation due to repeated passage throughgraded alcohols can cause suboptimal results. In cases withonly 1 slide available and multiple markers to test, the sampleis divided into zones and a different antibody is applied to eachzone; alternatively, the sample can be divided using tissuetransfer techniques.329 Cell transfer techniques allow the eva-luation of multiple markers when few slides are available.86

If cell transfer from previously stained cytologic smears isanticipated, nonadhesive treated slides should be used.239

Cytologic Slide Storage. Storage of air-dried preparations for up to2 weeks at 2!C to 8!C before ICC does not appear to reducetheir antigenicity.100 If longer storage is needed, slides shouldbe kept at –70!C.345,362 Some laboratories fix cytologicsamples with methanol and, if not used immediately, cover themwith 3% polyethylene glycol for storage or transportation.188

Fixation and Antigen Retrieval. Another difference between IHCand ICC is the type of fixation. In contrast to IHC, in which10% formalin is used routinely, there is no standard fixative forcytologic specimens. In general, the type of fixation is deter-mined by the cytological procedure and the antigens to betested.345 In ICC, samples are wet-fixed or air-dried and fixedimmediately before performing ICC with 100% acetone or10% formalin.73,379 Air-dried preparations tend to lose fewercells than wet-fixed preparations, but inconsistent ICC resultshave been reported with air-dried samples.86 For nuclear anti-gens, fixation of air-dried specimens in buffered 4% to 10%formalin alone or followed by methanol-acetone producesexcellent results,345,361 although air-drying of smears beforefixation has been reported to hinder estrogen or progesteronereceptor detection.239 For membrane and cytoplasmic antigens,the type of fixation is not as critical as for nuclear antigens;various fixatives, including formalin followed by ethanol, a1:1 mixture of methanol-absolute ethanol, or acetone at –20!C, produce good results.345 However, acetone solubilizescell membranes, leading to diffusion of small peptides out ofcells and false-negative results (Fig. 6).383 Antigens such asS100 protein, Hep Par 1, and gross cystic fluid protein 15 areleached by alcohol fixatives, producing false-negativeresults.62 One laboratory proposes these guidelines: samplesshould be fixed immediately prior to the ICC procedure; forlymphoid and melanoma markers, samples should be fixed for5 to 10 minutes at RT in acetone; for epithelial markers, 5 min-utes at RT in 95% ethanol or 1:1 mixture of methanol and 100%ethanol; and for nuclear antigens, 3.7% buffered formalin for




15 minutes.100 Formal saline has been proposed as a ‘‘univer-sal’’ ICC fixative.207 Others advocate air-drying slides if alymphoma is suspected and immediately fixing smears in95% ethanol (without air-drying) in all other cases.239 A recentcomparison of FFPE cell blocks (CBs) with alcohol-fixedcentrifuged preparations (AFCPs) did not find significant dif-ferences.166 Cytologic preparations fixed in acetone may bemore susceptible to cell loss than those fixed in formalin whenusing automatic stainers because of weaker cytoplasmic attach-ment to the glass slides.

AFCPs without AR performed similarly to cell blocks withAR in many cases; however, for some tests, particularly to detectnuclear antigens, AR has improved the reactivity of AFCPs.166

HIER using citrate buffer pH 6.0 is necessary for most nuclearantigens on ethanol-fixed cytologic smears; for some cytoplas-mic membrane and cytoplasmic antigens, it enhances the immu-noreaction.80 Few cytoplasmic membrane antigens requireHIER with higher pH buffers.80 Some laboratories performHIER irrespective of the fixation type.435 Enzymatic AR is muchless commonly used than HIER in ICC.435 The mechanism ofaction of HIER might depend on the fixative. When the amountof cytologic specimen is limited, the same slide can be tested fora second marker if the first test is negative.74 Adhesive slidesshould be used to perform HIER or protease AR on smears; oth-erwise, the sample is likely to detach.239

Immunocytochemical Method. Immunocytochemical proceduresare similar to those for IHC.286 Endogenous peroxidase is blockedwith 3% hydrogen peroxide; this step is unnecessary in acetone-fixed samples. In blood-rich smears, the use of alkalinephosphatase procedures rather than immunoperoxidase avoidsthe background produced by endogenous peroxidase without theneed for strong quenching reagents.86 Antigen retrieval is oftennecessary even without formalin fixation. Unfortunately, thewide range of fixation procedures makes interlaboratorystandardization of AR difficult; each laboratory must optimize theprocedure for each antigen.188,286,341 Remarkably, in a multi-institutional study, IHC quality was good irrespective of thecytologic preparation or fixation procedure, except that cell blockpreparations consistently had the highest scores.188

Controls and Standardization in Immunocytochemistry. Otherchallenges in ICC are (1) use of adequate positive and negativecontrols (ideally, controls should be treated in the same way astest samples), (2) nonstandardized methodology and antibodytiters, (3) difficulty in evaluation of immunoreactivity ininflamed or necrotic samples, (4) difficulty in distinguishingnormal from neoplastic cells, and (5) deleterious effect ofprotease digestion in non–formalin-fixed material.107,188

Positive and negative controls are standardized in humanand veterinary IHC284,286 and should be included with each testsample in ICC.62 Although the ideal tissue control is a compar-ably fixed cytologic preparation,62 only 13% of publicationslisted positive and negative controls processed identically asthe samples; 54% did not mention the use of controls or pro-cessed controls separately.64 The College of American

Pathologists recognizes the impracticality of maintainingseparate positive control samples for every possible combina-tion of fixation, processing, and specimen type (see commentfor questions ANP 22550 in the Anatomic Pathology checklistat http://www.cap.org/apps/cap.portal). Cytologic control pre-parations fixed in acetone lose antigenicity after several monthseven if wrapped in aluminum foil and refrigerated; controlsamples fixed in formalin retain their antigenicity indefi-nitely.379 The production of cytologic controls from organs(eg, lymph node, liver) is described elsewhere.379

Interpretation. As in IHC, the interpretation of ICC requiresconsideration of the ‘‘antibody personality profile’’ and the‘‘infidelity’’ (cross-reaction with antigens in other cell types)of tumor-specific markers.425 First, a positive or negativereaction must be defined.286 With the general lack of clearguidelines, each laboratory should document and state in theICC report what is interpreted as a positive result. Alterna-tively, as recommended in human IHC,124 a statement in theICC report indicating the intensity of the labeling and percent-age of positive cells (of the type of interest) is more informativethan just a positive versus negative result. As for IHC, interpre-tation of ICC tests should be done in conjunction with astandard cytologic stain (eg, Wright-Giemsa, Papanicolaou)and in concert with the clinicopathologic correlations.62

Causes of false-positive results in ICC include inadequatefixation, insufficient blocking of endogenous peroxidase orbiotin activities, and high background labeling with PAbs, ordetection of immunoreactivity in the wrong cell type (eg, nor-mal vs neoplastic).62,239,345 Two other causes of false-positiveresults are spurious staining of cells that have phagocytizedother cells and reagent trapping in clusters or layers of cells.239

False-negative results are caused by improper fixation,inadequate antibody titration, insufficient AR, or cell damageduring preparation.62,345

The Future of Immunohistochemistry

Since IHC was developed in the 1940s,65 major advances havebeen made in the sensitivity and specificity of the method andthe availability of biomarkers for FFPE tissues. The main pur-pose of IHC has been to characterize a neoplasm or identify aninfectious agent. However, the current trend is to apply IHC tothe detection of genetic abnormalities in neoplasms, detectfindings of prognostic importance, and aid in selection ofagents for cancer treatment.208 These topics are actively inves-tigated in human medicine and not without controversy. Beforeapplying IHC to such investigations, the entire process must bestandardized, from the time a sample is taken from the patient(preanalytical variables) through the analytical steps andending with the IHC results and diagnosis (postanalytical vari-ables).|| Standardization of IHC procedures will be necessary tocompare results among laboratories and will be mandatory if

||References 10, 85, 208, 223, 308, 343, 369.




computer-assisted quantification is intended, particularly ifthose results influence the therapeutic approach. Although thebarriers to progress are daunting, the in situ detection of proteinexpression by IHC will be a vital part of pathology for years tocome, even if the generation, interpretation, and application oftest results in the context of disease change drastically.

Acknowledgements

We thank Marilyn Beissenherz, VMDL–University of Missouri, and DeeDuSold, ADDL–Purdue University, for their dedication to preparingIHC slides and troubleshooting. We also thank Dr Chang Kim, Compara-tive Pathobiology, Purdue University, West Lafayette, Indiana, and Drvan der Loos, Academic Medical Center, Amsterdam, the Netherlands,for reviewing portions of this manuscript. Finally, we thank the veterinar-ians who submitted cases for IHC to the laboratories in which the authorshave worked and learned the red, brown, and blue technique.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect tothe research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, author-ship, and/or publication of this article.

References

1. Abendroth CS, Dabbs DJ. Immunocytochemical staining of

unstained versus previously stained cytologic preparations. Acta

Cytol. 1995;39(3):379–386.

2. Absolom DR, van Oss CJ. The nature of the antigen-antibody

bond and the factors affecting its association and dissociation.

Crit Rev Immunol. 1986;6(1):1–46.

3. Adams JC. Biotin amplification of biotin and horseradish perox-

idase signals in histochemical stains. J Histochem Cytochem.

1992;40(10):1457–1463.

4. Alelu-Paz R, Iturrieta-Zuazo I, Byne W, et al. A new antigen retrie-

val technique for human brain tissue. PloS One. 2008;3(10):e3378.

5. Apple S, Pucci R, Lowe AC, et al. The effect of delay in fixation,

different fixatives, and duration of fixation in estrogen and pro-

gesterone receptor results in breast carcinoma. Am J Clin Pathol.

2011;135(4):592–598.

6. Arber DA. Effect of prolonged formalin fixation on the immuno-

histochemical reactivity of breast markers. Appl Immunohisto-

chem Mol Morphol. 2002;10(2):183–186.

7. Arber JM, Arber DA, Jenkins KA, et al. Effect of decalcification

and fixation in paraffin-section immunohistochemistry. Appl

Immunohistochem. 1996;4:241–248.

8. Arnold MM, Srivastava S, Fredenburgh J, et al. Effects of fixation

and tissue processing on immunohistochemical demonstration of

specific antigens. Biotech Histochem. 1996;71(5):224–230.

9. Athanasou NA, Quinn J, Heryet A, et al. Effect of decalcification

agents on immunoreactivity of cellular antigens. J Clin Pathol.

1987;40(8):874–878.

10. Baker AF, Dragovich T, Ihle NT, Williams R. Stability of

phosphoprotein as a biological marker of tumor signaling. Clin

Cancer Res. 2005;11(12):4338–4340.

11. Balaton AJ, Drachenberg CB, Rucker C, et al. Satisfactory

performance of primary antibodies beyond manufacturer’s

recommended expiration dates. Appl Immunohistochem Mol

Morphol. 1999;7(3):221–225.

12. Banerjee D, Pettit S. Endogenous avidin-binding activity in

human lymphoid tissue. J Clin Pathol. 1984;37(2):223–225.

13. Bankfalvi A, Navabi H, Bier B, et al. Wet autoclave pretreatment

for antigen retrieval in diagnostic immunohistochemistry. J

Pathol. 1994;174(3):223–228.

14. Barr NJ, Wu NCY. Cytopathology/FNA. In: Taylor CR, Cote RJ,

eds. Immunomicroscopy: A Diagnostic Tool for the Surgical

Pathologist. 3rd ed. Philadelphia, PA: Saunders; 2006:397–416.

15. Baszler TV, Evermann JF, Kaylor PS, et al. Diagnosis of naturally

occurring bovine viral diarrhea virus infections in ruminants using

monoclonal antibody–based immunohistochemistry. Vet Pathol.

1995;32(6):609–618.

16. Baszler TV, Kiupel M, Williams ES, et al. Comparison of two auto-

mated immunohistochemical procedures for the diagnosis of scrapie

in domestic sheep and chronic wasting disease in North American

white-tailed deer (Odocoileus virginianus) and mule deer (Odocoi-

leus hemionus). J Vet Diagn Invest. 2006;18(2):147–155.

17. Battifora H. The multi-tumor (sausage) tissue block: novel

method for immunohistochemical antibody testing. Lab Invest.

1986;55(2):244–248.

18. Battifora H. Assessment of antigen damage in immunohistochemis-

try: the vimentin internal control. Am J Clin Pathol. 1991;96(5):

669–671.

19. Battifora H. Quality assurance issues in immunohistochemistry.

J Histotechnol. 1999;22(3):169–175.

20. Battifora H, Kopinski M. The influence of protease digestion and

duration of fixation on the immunostaining of keratins. J

Histochem Cytochem. 1986;34(8):1095–1100.

21. Belloni B, Lambertini C, Nuciforo P, et al. Will PAXgene substitute

formalin? A morphological and molecular comparative study using

a new fixative system. J Clin Pathol. 2013;66(2):124–135.

22. Benavides J, Garcıa-Pariente C, Gelmetti D, et al. Effects of fixa-

tive type and fixation time on the detection of Maedi Visna virus

by PCR and immunohistochemistry in paraffin-embedded ovine

lung samples. J Virol Methods. 2006;137(2):317–324.

23. Bendayan M. Possibilities of false immunocytochemical results

generated by the use of monoclonal antibodies: the example of the

anti-proinsulin antibody. J Histochem Cytochem. 1995;43(9):

881–886.

24. Bergin IL, Smedley RC, Esplin DG, et al. Prognostic evaluation

of Ki67 threshold value in canine oral melanoma. Vet Pathol.

2011;48(1):41–53.

25. Berglund L, Bjorling E, Oksvold P, et al. A genecentric human

protein atlas for expression profiles based on antibodies. Mol Cell

Proteom. 2008;7(10):2019–2027.

26. Bertheau P, Cazals-Hatem D, Meignin V, et al. Variability of

immunohistochemical reactivity on stored paraffin slides. J Clin

Pathol. 1998;51(5):370–374.

27. Bian Y-S, Osterheld M-C, Bosman FT, et al. Nuclear accumula-

tion of beta-catenin is a common and early event during neoplas-

tic progression of Barrett esophagus. Am J Clin Pathol. 2000;

114(4):583–590.




28. Bilous M, Dowsett M, Hanna W, et al. Current perspectives on

HER2 testing: a review of national testing guidelines. Mod

Pathol. 2003;16(2):173–182.

29. Binder CJ, Horkko S, Dewan A, et al. Pneumococcal vaccination

decreases atherosclerotic lesion formation: molecular mimicry

between Streptococcus pneumoniae and oxidized LDL. Nat Med.

2003;9(6):736–743.

30. Blind C, Koepenik A, Pacyna-Gengelbach, et al. Antigenicity

testing by immunohistochemistry after tissue oxidation. J Clin

Pathol. 2008;61(1):79–83.

31. Boenisch T. Diluent buffer ions and pH: their influence on the

performance of monoclonal antibodies in immunohistochemistry.

Appl Immunohistochem Mol Morphol. 1999;7(4):300–306.

32. Boenisch T. Handbook on Immunohistochemical Staining Meth-

ods. 3rd ed. Carpinteria, CA: DAKO Corporation; 2001.

33. Boenisch T. Formalin-fixed and heat-retrieved tissue antigens: a

comparison of their immunoreactivity in experimental antibody

diluents. Appl. Immunohistochem Mol Morphol. 2001;9(2):

176–179.

34. Boenisch T. Heat-induced antigen retrieval restores electrostatic

forces: prolonging the antibody incubation as an alternative. Appl

Immunohistochem Mol Morphol. 2002;10(4):363–367.

35. Boenisch T. Effect of heat-induced antigen retrieval following

inconsistent formalin fixation. Appl Immunohistochem Mol

Morphol. 2005;13(3):283–286.

36. Boenisch T. Heat-induced antigen retrieval: what are we retriev-

ing? J Histochem Cytochem. 2006;54(9):961–964.

37. Boenisch T. Basic immunohistochemistry. In: Key M, ed. Immu-

nohistochemical Staining Methods. 4th ed. Carpinteria, CA: Dako

Corporation; 2006:15–25.

38. Bogen SA, Vani K, McGraw B, et al. Experimental validation of

peptide immunohistochemistry controls. Appl Immunohistochem

Mol Morphol. 2009;17(3):239–246.

39. Bogen SA, Vani K, Sompuram SR. Molecular mechanisms of

antigen retrieval: antigen retrieval reverses steric interference

caused by formalin-induced cross-links. Biotech Histochem.

2009;84(5):207–215.

40. Bordeaux J, Welsh AW, Agarwal S, et al. Antibody validation.

Biotechniques. 2010;48(3):197–209.

41. Bork P, Doolittle RF. Proposed acquisition of an animal protein

domain by bacteria. Proc Natl Acad Sci U S A. 1992;89(19):

8990–8994.

42. Bos PK, van Osch GJ, van der Kwast T, et al. Fixation-

dependent immunolocalization shift and immunoreactivity of

intracellular growth factors in cartilage. Histochem J. 2000;

32(7):391–396.

43. Bratthauer GL. Processing of tissue culture cells. In: Oliver C,

Jamur MC, eds. Immunocytochemical Methods and Protocols.

New York, NY: Humana Press; 2010:85–92.

44. Breitbart RE, Andreadis A, Nadal-Ginard B. Alternative spli-

cing: a ubiquitous mechanism for the generation of multiple pro-

tein isoforms from single genes. Ann Rev Biochem. 1987;56:

467–495.

45. Brown RW. Immunocytochemistry. In: Ramzy I, ed. Clinical

Cytopathology and Aspiration Biopsy: Fundamental Principles

and Practice. New York, NY: McGraw-Hill; 2001:535–548.

46. Buchwallow IB, Bocker W. Antibodies for immunohistochem-

istry. In: Buchwallow I, Bocker W, eds. Immunohistochemis-

try: Basics and Methods. Berlin, Germany: Springer-Verlag;

2010:1–8.

47. Buchwallow IB, Bocker W. Working with antibodies. In: Buch-

wallow I, Bocker W, eds. Immunohistochemistry: Basics and

Methods. Berlin, Germany: Springer-Verlag; 2010:31–39.

48. Buchwallow IB, Bocker W. Immunostaining enhancement. In:

Buchwallow I, Bocker W, eds. Immunohistochemistry: Basics

and Methods. Berlin, Germany: Springer-Verlag; 2010:47–59.

49. Buchwallow IB, Bocker W. Antigen detection on tissues using

primary antibody raised in the same species. In: Buchwallow I,

Bocker W, eds. Immunohistochemistry: Basics and Methods.

Berlin, Germany: Springer-Verlag; 2010:77–81.

50. Buchwalow I, Somoloiva V, Boecker W, Tiemann M. Non-

specific binding of antibodies in immunohistochemistry: fallacies

and facts. Sci Rep. 2011;1:28.

51. Buesa RJ. Histology without formalin? Ann Diagn Pathol. 2008;

12(6):387–396.

52. Buesa RJ, Peshkov MV. Histology without xylene. Ann Diagn

Pathol 2009;13(4):246–256.

53. Buesa RJ, Peshkov MV. How much formalin is enough to fix tis-

sues? Ann Diagn Pathol. 2012;16(3):202–209.

54. Burchette J, Zimmerman B. Antibody optimization and valida-

tion. Histologic. 2010;43(2):29–33.

55. Burry RW. Controls for immunocytochemistry: an update. J His-

tochem Cytochem. 2011;59(1):6–12.

56. Bussolati G, Gugliotta P, Volante M, et al. Retrieved endogen-

ous biotin: a novel marker and a potential pitfall in diagnostic

immunohistochemistry. Histopathology. 1997;31(5):400–407.

57. Cano G, Milanezi F, Leitao D, et al. Estimation of hormone recep-

tor status in fine-needle aspirates and paraffin-embedded sections

from breast cancer using the novel rabbit monoclonal antibodies

SP1 and SP2. Diagn Cytol. 2003;29(4):207–211.

58. Cao Q, Liu F, Xiao P, et al. Cytoplasmic staining of TTF-1 anti-

body in the diagnosis of hepatocellular carcinoma: study on 299

cases using tissue microarray. Pathology. 2011;2011:257352.

59. Cardiff RD, Hubbard NE, Engelberg JA, et al. Quantitation of

fixative-induced morphologic and antigenic variation in mouse

and human breast cancers. Lab Invest. 2013;93(4):480–497.

60. Chen B-X, Szabolcs MJ, Matsushima AY, Erlanger BF. A strategy

for immunohistochemical signal enhancement by end-product

amplification. J Histochem Cytochem. 1996;44(8):819–824.

61. Chen C, Sun SR, Gong YP, et al. Quantum dots–based molecular

classification of breast cancer by quantitative spectroanalysis of

hormone receptors and HER2. Biomaterials. 2011;32(30):

7592–7599.

62. Chivukula M, Dabbs DJ. Immunocytology. In: Dabbs DJ, ed.

Diagnostic Immunocytochemistry. 3rd ed. New York: Churchill

Livingstone; 2010:890–918.

63. Cohn M. Degeneracy, mimicry and crossreactivity in immune

recognition. Mol Immunol. 2005;42(5):651–655.

64. Colasacco C, Mount S, Leiman G. Documentation of immunocy-

tochemistry controls in the cytopathologic literature: a meta-

analysis of 100 journal articles. Diagn Cytopathol. 2010;39(4):

245–250.




65. Coons AH, Creech HJ, Jones RN. Immunological properties of an

antibody containing a fluorescent group. Proc Soc Exp Biol Med.

1941;47(2):200–202.

66. Coons AH, Kaplan MH. Localization of antigen in tissue cells, II:

improvements in a method for the detection of antigen by means

of fluorescent antibody. J Exp Med. 1950;91(1):1–13.

67. Coons AH, Leduc EH, Connolly JM. Studies on antibody produc-

tion, I: a method for the histochemical demonstration of specific

antibody and its application to a study of the hyperimmune rabbit.

J Exp Med. 1955;102(1):49–60.

68. Copete M, Garratt J, Gilks B, et al. Inappropriate calibration and

optimization of pan-keratin (pan-CK) and low molecular weight

keratin (LMWCK) immunohistochemistry tests: Canadian Immu-

nohistochemistry Quality Control (CIQC) experience. J Clin

Pathol. 2011;64(3):220–225.

69. Couchman JR. Commercial antibodies: the good, bad, and really

ugly. J Histochem Cytochem. 2009;57(1):7–8.

70. Cross SS, Start RD, Smith JHF. Does delay in fixation affect the

number of mitotic figures in processed tissue? J Clin Pathol.

1990;43(7):597–599.

71. Cummings J, Raynaud F, Jones L, et al. Fit-for-purpose biomarker

method validation for application in clinical trials of anticancer

drugs. Br J Cancer. 2010;103(9):1313–1317.

72. Cunha-Neto E, Bilate AM, Hyland KV, et al. Induction of cardiac

autoimmunity in Chagas heart disease: a case for molecular mimi-

cry. Autoimmunity 2006;39(1):41–54.

73. Dabbs DJ. Immunocytology. In: Dabbs DJ, ed. Diagnostic Immu-

nocytochemistry. 2nd ed. New York: Churchill Livingstone;

2002:625–639.

74. Dabbs DJ, Wang X. Immunocytochemistry on cytologic speci-

mens of limited quantity. Diagn Cytopathol. 1998;18(2):166–169.

75. Dapson RW. Fixation for the 1990’s: a review of needs and

accomplishments. Biotech Histochem. 1993;68(2):75–82.

76. Dapson RW. Glyoxal fixation: how it works and why it only occa-

sionally needs antigen retrieval. Biotech Histochem. 2007;82(3):

161–166.

77. Dapson RW. Mechanisms of action and proper use of common

fixatives. In: Shi S-R, Taylor CR, eds. Antigen Retrieval Immuno-

histochemistry Based Research and Diagnostics. Hoboken, NJ:

John Wiley; 2010:195–217.

78. Davidoff M, Schulze W. Combination of the peroxidase anti-

peroxidase (PAP) and avidin-biotin-peroxidase complex

(ABC)–techniques: an amplification alternative in immunocyto-

chemical staining. Histochemistry. 1990;93(5):531–536.

79. De Cecco L, Musella V, Veneroni S, et al. Impact of biospeci-

mens handling on biomarker research in breast cancer. BMC Can-

cer. 2009;9:409.

80. Denda T, Kamoshida S, Kawamura J, et al. Optimal antigen

retrieval for ethanol-fixed cytologic smears. Cancer Cytopathol.

2012;120(3):167–176.

81. Di Vito KA, Charette LA, Rimm DL, et al. Long-term preserva-

tion of antigenicity on tissue microarrays. Lab Invest. 2004;84(8):

1071–1078.

82. Donhuijsen K, Schmidt U, Hirche H, et al. Changes in mitotic rate

and cell cycle fractions caused by delayed fixation. Hum Pathol.

1990;21(7):709–714.

83. Dotti I, Bonin S, Basili G, et al. Formalin-free fixatives. In: Stanta

G, ed. Guidelines for Molecular Analysis in Archive Tissues.

Berlin, Germany: Springer-Verlag; 2011:13–18.

84. Duhamel RC, Johnson DA. Use of nonfat dry milk to block non-

specific nuclear and membrane staining by avidin conjugates. J


85. Dunstan RW, Wharton KA, Quigley C, et al. The use of immuno-

histochemistry for biomarker assessment: can it compete with

other technologies? Toxicol Pathol. 2011;39(6):988–1002.

86. Dupre MP, Courtade-Saidi M. Immunocytochemistry as an

adjunct to diagnostic cytology. Ann Pathol. 2012;32(6):433–437.

87. Ehara H, Deguchi T, Koji T, et al. Autoclave antigen retrieval

technique for immunohistochemical staining of androgen receptor

in formalin-fixed paraffin sections of human prostate. Acta Histo-

chem Cytochem. 1996;29(4):311–318.

88. Elias JM. Antigen restoration: the ‘‘hot’’ revolution in immuno-

histochemistry. J Histotechnol. 2001;24(3):193–198.

89. Elias JM. Immunohistochemical methods. In: Elias JM, ed. Immu-

nohistopathology: A Practical Approach to Diagnosis. 2nd ed.

Chicago, IL: ASCP Press; 2003:1–110.

90. Elias JM, Margiotta M, Gaborc D. Sensitivity and detection

efficiency of the peroxidase antiperoxidase (PAP), avidin-

biotin peroxidase complex (ABC), and peroxidase-labeled

avidin-biotin (LAB) methods. Am J Clin Pathol. 1989;92(1):

62–67.

91. Eltoum I, Fredenburgh J, Grizzle WE. Advanced concepts in fixa-

tion: 1. Effects of fixation on immunohistochemistry, reversibility

of fixation and recovery of proteins, nucleic acids, and other

molecules from fixed and processed tissues. 2. Developmental

methods of fixation. J Histotechnol. 2001;24(3):201–210.

92. Eltoum I, Fredenburgh J, Myers RB, et al. Introduction to the the-

ory and practice of fixation of tissues. J Histotechnol. 2001;24(3):

173–190.

93. Engel KB, Moore HM. Effects of preanalytical variables on the

detection of proteins by immunohistochemistry in formalin-

fixed, paraffin-embedded tissue. Arch Pathol Lab Med. 2011;

135(5):537–543.

94. Espina V, Mueller C, Edmiston K, et al. Tissue is alive: new tech-

nologies are needed to address the problems of protein biomarker

pre-analytical variability. Proteomics Clin Appl. 2009;3(8):

874–882.

95. Eusebi V, Bondi A, Rosai J. Immunohistochemical localization of

myoglobin in nonmuscular cells. Am J Surg Pathol. 1984;8(1):

51–55.

96. Ewing-Finchem T. Effects of fixation and decalcification on

kappa and lambda staining by immunohistochemistry. Histologic.

2008;41(1):1–4.

97. Ezaki T. Antigen retrieval on formaldehyde-fixed paraffin

sections: its potential drawbacks and optimization for double

immunostaining. Micron. 2000;31(6):639–649.

98. Fang M, Peng C-W, Pang D-W, Li Y. Quantum dots for cancer

research: current status, remaining issues, and future perspectives.

Cancer Biol Med. 2012;9(3):151–163.

99. Faoro V, Sapino A. Tissue microarray (TMA). In: Stanta G, ed.

Guidelines for Molecular Analysis in Archive Tissues. Berlin,

Germany: Springer-Verlag; 2011:23–26.




100. Fetsch PA, Abati A. Ancillary techniques in cytopathology. In:

Atkinson BF, ed. Atlas of Diagnostic Cytopathology. 2nd ed.

Philadelphia, PA: Saunders; 2004:747–775.

101. Fetsch PA, Abati A. The clinical immunohistochemistry labora-

tory: regulations and troubleshooting guidelines. Methods Mol

Biol. 2010;588:399–412.

102. Fiore C, Bailey D, Conlon N, et al. Utility of multispectral

imaging in automated quantitative scoring of immunohisto-

chemistry. J Clin Pathol. 2012;65(6):496–502.

103. Fitzgerald SD, Richard A. Comparison of four fixatives for

routine splenic histology and immunohistochemical staining for

group II avian adenovirus. Avian Dis. 1995;39(2):425–431.

104. Fletcher CDM, Fletcher JA. Testing for KIT (CD117) in gastro-

intestinal stromal tumors: another Hercep test? Am J Clin Pathol.

2002;118(2):163–164.

105. Floyd AD. Image analysis in immunohistochemistry. In: Shi

S-R, Taylor CR, eds. Antigen Retrieval Immunohistochemistry

Based Research and Diagnostics. Hoboken, NJ: John Wiley;

2010:165–185.

106. Floyd L, van der Loos CM. The 2011 approach: how to make a

new antibody work for IHC, and characterize the staining pat-

tern? Workshop #54. Paper presented at: 37th NSH Convention;

September 16–21, 2011; Cincinnati, OH.

107. Fowler LJ, Lachar WA. Application of immunohistochemistry

to cytology. Arch Pathol Lab Med. 2008;132(3):373–383.

108. Fowler CB, O’Leary TJ, Mason JT. Modeling formalin fixation

and histological processing with ribonuclease A: effects of etha-

nol dehydration on reversal of formaldehyde cross-links. Lab

Invest. 2008;88(7):785–791.

109. Fox CH, Johnson FB, Whiting J, et al. Formaldehyde fixation. J


110. Fraenkel-Conrat H, Brandon BA, Olcott HS. The reaction of for-

maldehyde with proteins, IV: participation of indole groups.

Gramicidin. J Biol Chem. 1947;168:99–118.

111. Fraenkel-Conrat H, Olcott HS. The reaction of formaldehyde with

proteins, V: cross-linking between amino and primary amide or

guanidyl groups. J Am Chem Soc. 1948;70:2673–2684.

112. Fraenkel-Conrat H, Olcott HS. Reaction of formaldehyde with

proteins, VI: cross-linking of amino groups with phenol, imida-

zole, or indole groups. J Biol Chem. 1948;174:827–843.

113. Fredenburgh JL, Myers RB. Basic IHC workshop. Presented at:

28th Annual Meeting of the National Society for Histotechnol-

ogy; September 29-October 3, 2002; Long Beach, CA.

114. Freeland J, von Bueren E, Muralitharan S, et al. Water-soluble

organic solvent improves paraffin displacement in all-in-one

epitope retrieval buffers. J Histotechnol. 2013;36(2):51–58.

115. Fritz K. Method for making multitissue control blocks. J Histo-

technol. 2010;33(1):49–51.

116. Fung K-M, Messing A, Lee VMY, et al. A novel modification of

the avidin-biotin complex method for immunohistochemical

studies of transgenic mice with murine monoclonal antibodies.

J Histochem Cytochem. 1992;40(9):1319–1328.

117. Furuya T, Ikemoto K, Kawauchi S, et al. A novel technology

allowing immunohistochemical staining of a tissue section with

50 different antibodies in a single experiment. J Histochem Cyto-

chem. 2004;52(2):205–210.

118. Gatta LB, Cadel M, Balzarini P, et al. Application of alternative

fixatives to formalin in diagnostic pathology. Eur J Histochem.

2012;56(2):e12.

119. Gillett CE, Springall RJ, Bames DM, et al. Multiple tissue core

arrays in histopathology research: a validation study. 2000;

192(4):549–553.

120. Giltnane JM, Rimm DL. Technology insight: identification of

biomarkers with tissue microarray technology. Nat Clin Pract

Oncol. 2004;1(2):104–111.

121. Giordano TJ. Gene expression profiling of endocrine tumors

using DNA microarrays: progress and promise. Endocr Pathol.

2003;14(2):107–116.

122. Glass G, Papin JA, Mandell JW. SIMPLE: a sequential immuno-

peroxidase labeling and erasing method. J Histochem Cytochem.

2009;57(10):899–905.

123. Goldstein NS, Ferkowicz M, Odish W, et al. Minimum formalin

fixation time for consistent estrogen receptor immunohisto-

chemical staining of invasive breast carcinoma. Am J Clin

Pathol. 2003;120(1):86–92.

124. Goldstein NS, Hewitt SM, Taylor CR, et al. Recommenda-

tions for improved standardization of immunocytochemis-

try. Appl Immunohistochem Mol Morphol. 2007;15(2):

124–133.

125. Gottwald L, Sek P, Piekarski J, et al. Construction of a tissue

microarray with two millimeters cores of endometroid endome-

trial cancer: factors affecting the quality of the recipient block.

Biotech Biochem. 2012;87(8):512–518.

126. Gown AM. Modern immunohistochemistry in targeted therapy.

In: Cheng L, Zhang DY, Eble JN, eds. Molecular Genetic

Pathology. New York, NY: Springer; 2013:181–196.

127. Grabau DA, Nielsen O, Hansen S, et al. Influence of storage

temperature and high-temperature antigen retrieval buffers on

results of immunohistochemical staining in sections stored

for long periods. Appl Immunohistochem. 1998;6(4):209–213.

128. Grizzle WE. Special symposium: fixation and tissue processing

models. Biotech Histochem. 2009;84(5):185–193.

129. Grizzle WE, Stockard CR, Billings PE. The effects of tissue pro-

cessing variables other than fixation on histochemical staining

and immunohistochemical detection of antigens. J Histotechnol.

2001;24(3):213–219.

130. Groelz D, Sobin L, Branton P, et al. Non-formalin fixative ver-

sus formalin-fixed tissues: a comparison of histology and RNA

quality. Exp Mol Pathol. 2013;94:188–194.

131. Gross AJ, Sizer IW. The oxidation of tyramine, tyrosine, and

related compounds by peroxidase. J Biol Chem. 1959;234(6):

1611–1614.

132. Groves DJ, Morris BA. Veterinary sources of nonrodent mono-

clonal antibodies: interspecific and intraspecific hybridomas.

Hybridoma. 2000;19(3):201–214.

133. Guerrero RB, Batts KP, Brandhagen DJ, et al. Effects of forma-

lin fixation and prolonged block storage on detection of hepatitis

C virus RNA in liver tissue. Diagn Mol Pathol. 1997;6(5):

277–281.

134. Guesdon JL, Ternynck T, Avrameas S. The uses of avidin-biotin

interaction in immunoenzymatic techniques. J Histochem Cyto-

chem. 1979;27(8):1131–1139.




135. Guo T, Wang W, Rudnick PA, et al. Proteome analysis of

microdissected formalin-fixed and paraffin-embedded tissue

specimens. J Histochem Cytochem. 2007;55(7):763–772.

136. Hammond MEH, Hayes DF, Dowsett M, et al. American Society

of Clinical Oncology/College of American Pathologists

guideline recommendations for immunohistochemical testing

of estrogen and progesterone receptors in breast cancer (unab-

ridged version). Arch Pathol Lab Med. 2010;134(7):e48–e72.

137. Hansen BL, Winther H, Moller K. Excessive section drying of

breast cancer tissue prior to deparaffinization and antigen retrie-

val causes a loss in HER2 immuno-reactivity. Immunocytochem-

istry. 2008;6(3):119–122.

138. Harley R, Gruffydd-Jones TJ, Day MJ. Non-specific labelling of

mast cells in feline oral mucosa: a potential problem in immuno-

histochemical studies. J Comp Pathol. 2002;127(2–3):228–231.

139. Hasui K, Murata F. A new simplified catalyzed signal amplifica-

tion system for minimizing non-specific staining in tissues with

supersensitive immunohistochemistry. Arch Histol Cytol. 2005;

68(1):1–17.

140. Hasui K, Takatsuka T, Sakamoto R, et al. Improvement of super-

sensitive immunohistochemistry with an autostainer: a simpli-

fied catalyzed signal amplification system. Histochem J. 2002;

34(5):215–222.

141. Hayat MA. Antigens and antibodies. In: Hayat MA, ed. Micro-

scopy, Immunohistochemistry, and Antigen Retrieval Methods

for Light and Electron Microscopy. New York, NY: Kluwer

Academic; 2002:31–51.

142. Hayat MA. Fixation and embedding. In: Hayat MA, ed. Micro-

scopy, Immunohistochemistry, and Antigen Retrieval Methods

for Light and Electron Microscopy. New York, NY: Kluwer

Academic; 2002:71–93.

143. Hayat MA. Factors affecting antigen retrieval. In: Hayat MA, ed.

Microscopy, Immunohistochemistry, and Antigen Retrieval

Methods for Light and Electron Microscopy. New York, NY:

Kluwer Academic; 2002:53–69.

144. Hayat MA. Antigen retrieval. In: Hayat MA, ed. Microscopy,

Immunohistochemistry, and Antigen Retrieval Methods for Light

and Electron Microscopy. New York, NY: Kluwer Academic;

2002:117–139.

145. Helander KG. Kinetic studies of formaldehyde binding in tissue.

Biotech Histochem. 1994;69(3):177–179.

146. Henshall S. Tissue microarrays. J Mammary Gland Biol Neopl.

2003;8(3):347–358.

147. Henwood AF. Effect of slide drying at 80 C on immunohisto-

chemistry. J Histotechnol. 2005;28(1):45–46.

148. Henwood AF. The application of heated detergent dewaxing and

rehydration to immunohistochemistry. Biotech Histochem.

2012;87(1):46–50.

149. Hewitt SM, Robinowitz M, Bogen SA, et al. Quality Assurance

for Design Control and Implementation of Immunohistochemis-

try Assays: Approved Guideline. 2nd ed. CLSI document I/

LA28-A2. Wayne, PA: Clinical and Laboratory Standards Insti-

tute; 2011.

150. Hewitt SM, Takikita M, Abedi-Ardekani B, et al. Validation of

proteomics based discovery with tissue microarrays. Proteom

Clin Appl. 2008;2(10–11):1460–1466.

151. Hicks DG, Boyce BF. The challenge and importance of standar-

dizing pre-analytical variables in surgical pathology specimens

for clinical care and translational research. Biotech Histochem.

2012;87(1):14–17.

152. Hierck BP, Iperen LV, Gittenberger-de Groot AC, et al.

Modified indirect immunodetection allows study of murine tis-

sue with mouse monoclonal antibodies. J Histochem Cytochem.

1994;42(11):1499–1502.

153. Hoetelmans RWM, Prins FA, Velde IC, et al. Effects of acetone,

methanol, or paraformaldehyde on cellular structure, visualized

by reflection contrast microscopy and transmission and scanning

electron microscopy. Appl Immunohistochem Mol Morphol.

2001;9(4):346–351.

154. Hoetelmans RWM, van Slooten H-J, Keijzer R, et al. Routine for-

maldehyde fixation irreversibly reduces immunoreactivity of Bcl-

2 in the nuclear compartment of breast cancer cells, but not in the

cytoplasm. Appl Immunohistochem Mol Morphol. 2001;9(1):74–80.

155. Holmseth S, Zhou Y, Follin-Arbelet VV, et al. Specificity con-

trols for immunocytochemistry: the antigen preadsorption test

can lead to inaccurate assessment of antibody specificity. J His-

tochem Cytochem. 2012;60(3):174–187.

156. Hornick JL, Fletcher CDM. Immunohistochemical staining for

KIT (CD 117) in soft tissue sarcomas is very limited in distribu-

tion. Am J Clin Pathol. 2002;117(2):188–193.

157. Hsi ED. A practical approach for evaluating new antibodies in

the clinical immunohistochemistry laboratory. Arch Pathol Lab

Med. 2001;125(2):289–294.

158. Hsu FD, Nielsen TO, Alkushi A, et al. Tissue microarrays are an

effective quality assurance tool for diagnostic immunohisto-

chemistry. Mod Pathol. 2002;15(12):1374–1380.

159. Hsu S-M, Raine L. Protein A, avidin and biotin in immunocyto-

chemistry. J Histochem Cytochem. 1981;29(11):1349–1353.

160. Hsu S-M, Raine L, Fanger H. Use of avidin-biotin-peroxidase

complex (ABC) in immunoperoxidase techniques: a comparison

between ABC and unlabeled antibody (PAP) procedures. J


161. Huang S-N. Immunohistochemical demonstration of hepatitis B

core and surface antigens in paraffin sections. Lab Invest. 1975;

33(1):88–95.

162. Huang S-N, Minassian H, More JD. Application of immuno-

fluorescent staining on paraffin sections improved by trypsin

digestion. Lab Invest. 1976;35(4):383–390.

163. Huang W, Hennrick K, Drew S. A colorful future of quantitative

pathology: validation of Vectra technology using chromogenic

multiplexed immunohistochemistry and prostate tissue microar-

rays. Hum Pathol. 2013;44(1):29–38.

164. Ibarra JA, Rogers LW. Fixation time does not affect expression of

HER2/neu: a pilot study. Am J Clin Pathol. 2010;134(4):594–596.

165. Iizuka M, Chiba M, Yukawa M, et al. Immunohistochemical anal-

ysis of the distribution of measles related antigen in the intestinal

mucosa in inflammatory bowel disease. Gut. 2000;46(2):163–169.

166. Ikeda K, Tate G, Suzuki T, et al. Comparison of immunocyto-

chemical sensitivity between formalin-fixed and alcohol-fixed

specimens reveals the diagnostic value of alcohol-fixed cytocen-

trifuged preparations in malignant effusion cytology. Am J Clin

Pathol. 2011;136(6):934–942.




167. Imam SA, Young L, Chaiwun B, et al. Comparison of two

microwave based antigen-retrieval solutions in unmasking

epitopes in formalin-fixed tissue for immunostaining. Antican-

cer Res. 1995;15(4):1153–1158.

168. International Organization for Standardization–International

Electrotechnical Commission. General Requirements for the

Competence of Testing and Calibration Laboratories. 2nd ed.

Geneva, Switzerland: International Organization for Standardi-

zation; 2005. ISO/ISE 17025.

169. Jacobs TW, Prioleau JE, Stillman IE, et al. Loss of tumor mar-

ker–immunostaining intensity on stored paraffin slides of breast

cancer. J Natl Cancer Inst. 1996;88(15):1054–1059.

170. Jacobsen M, Clausen PP, Smidth S. The effect of fixation and

trypsinization on the immunohistochemical demonstration of

intracellular immunoglobulin in paraffin embedded material.

Acta Path Microbiol Scand A. 1980;88(6):369–376.

171. James JD, Hauer-Jensen M. Effects of fixative and fixation time

for quantitative computerized image analysis of immunohisto-

chemical staining. J Histotechnol. 1999;22(2):109–111.

172. Jensen TA. Tissue microarray: advanced techniques. J Histo-

technol. 2003;26(2):101–104.

173. Jensen TA, Hammond MEH. The tissue microarray: a technical

guide for histologists. J Histotechnol. 2001;24(4):283–287.

174. Jin Z, Hildebrandt N. Semiconductor quantum dots for in vitro diag-

nostics and cellular imaging. Trends Biotech. 2012;30(7):394–403.

175. Johnson DA, Gautsch JW, Sportsman JR, et al. Improved tech-

nique utilizing nonfat dried milk for analysis of proteins and

nucleic acids transferred to nitrocellulose. Gene Anal Tech.

1984;1(1):3–8.

176. Jones ML. Lipids. In: Bancroft JD, Gamble M, eds. Theory and

Practice of Histological Techniques. 5th ed. Edinburg, UK:

Churchill-Livingstone; 2002:201–230.

177. Jones WT, Stockard CR, Grizzle WE. Effects of time and

temperature during attachment of sections to microscope slides

on immunohistochemical detection of antigens. Biotech Histo-

chem. 2001;76(2):55–58.

178. Josephsen K, Smith CE, Nanci A. Selective but nonspecific

immunolabeling of enamel protein–associated compartments

by a monoclonal antibody against vimentin. J Histochem

Cytochem. 1999;47(10):1237–1245.

179. Kai H, Shin R-W, Ogino K. Enhanced antigen retrieval of amy-

loid b immunohistochemistry: re-evaluation of amyloid bpathology in Alzheimer disease and its mouse model. J Histo-

chem Cytochem. 2012;60(10):761–769.

180. Kakimoto K, Takekoshi S, Miyajima K, et al. Hypothesis for the

mechanism for heat-induced antigen retrieval occurring on fresh

frozen sections without formalin-fixation in immunohistochem-

istry. J Mol Hist. 2008;39(4):389–399.

181. Kalyuzhny AE. The dark side of the immunohistochemical moon:

industry. J Histochem Cytochem. 2009;57(12):1099–1101.

182. Kanai K, Nunoya T, Shibuya K, et al. Variation in effectiveness

of antigen retrieval pretreatment for diagnostic immunohisto-

chemistry. Res Vet Sci. 1998;64(1):57–61.

183. Kap M, Smedts F, Oosterhuis W, et al. Histological assessment

of PAXgene tissue fixation and stabilization reagents. PLoS

One. 2011;6(11):e27704.

184. Kawaoi A, Okano T, Nemoto N, et al. Simultaneous detection of

thyroglobulin (Tg), thyroxine (T4) and triiodothyronine (T3) in

nontoxic thyroid tumors by the immunoperoxidase method.

Am J Pathol. 1982;108(1):39–49.

185. Keller SM, Moore PF. A novel clonality assay for the assessment

of canine T cell proliferations. Vet Immunol Immunopathol.

2012;145(1–2):410–419.

186. Khoury T, Sait S, Hwang H, et al. Delay to formalin fixation

effect on breast biomarkers. Mod Pathol. 2009;22(11):

1457–1467.

187. Kim DH, Jung KC, Shin YK, et al. The enhanced reactivity of

endogenous biotin-like molecules by antigen retrieval proce-

dures and signal amplification with tyramine. Histochem J.

2002;34(3–4):97–103.

188. Kirbis IS, Maxwell P, Flezar MS, et al. External quality control

for immunocytochemistry on cytology samples: a review of UK

NEQAS ICC (cytology module) results. Cytopathology. 2011;

22(4):230–237.

189. Kitamoto T, Ogomori K, Tateishi J, et al. Formic acid pretreat-

ment enhances immunostaining of cerebral and systemic amy-

loids. Lab Invest. 1987;57(2):230–236.

190. Kiupel M, Smedley RC, Pfent C, et al. Diagnostic algorithm to

differentiate lymphoma from inflammation in feline small intest-

inal biopsy samples. Vet Pathol. 2011;48(1):212–222.

191. Kiupel M, Webster JD, Kaneene JB, et al. The use of KIT

and tryptase expression patterns as prognostic tools for

canine cutaneous mast cell tumors. Vet Pathol. 2004;41(4):

371–377.

192. Klockenbusch C, O’Hara JE, Kast J. Advancing formaldehyde

cross-linking toward quantitative proteomic applications. Anal

Bioanal Chem. 2012;404(4):1057–1067.

193. Klopfleisch R, von Deetzen M, Weiss AT, et al. Weigners fixa-

tive: an alternative to formalin fixation for histology with

improved preservation of nucleic acids. Vet Pathol. 2013;

50(1):191–199.

194. Klopfleisch R, Weiss ATA, Gruber AD. Excavation of a buried

treasure: DNA, mRNA, miRNA and protein analysis in formalin

fixed, paraffin embedded tissues. Histol Histopathol. 2011;

26(6):797–810.

195. Koch S, Stappenbeck N, Cornelissen CG, et al. Tissue engineer-

ing: selecting the optimal fixative for immunohistochemistry.

Tissue Eng Part C Methods. 2012;18(12):976–983.

196. Kohler G, Milstein C. Derivation of specific antibody-producing

tissue culture and tumor lines by cell fusion. Eur J Immunol.

1976;6(7):511–519.

197. Kononen J, Bubendorf L, Kallioniemi A, et al. Tissue microar-

rays for high-throughput molecular profiling of tumor speci-

mens. Nat Med. 1998;4(7):844–847.

198. Kothmaier H, Rohrer D, Stacher E, et al. Comparison of

formalin-free tissue fixatives: a proteomic study testing their

application for routine pathology and research. Arch Pathol Lab

Med. 2011;135(6):744–752.

199. Krenacs L, Krenacs T, Stelkovics E, et al. Heat-induced antigen

retrieval for immunohistochemical reactions in routinely pro-

cessed paraffin sections. Methods Mol Biol. 2010;588:

103–119.




200. Kurien BT, Dorri Y, Dillon S, et al. An overview of Western

blotting for determining antibody specificities for immunohisto-

chemistry. Methods Mol Biol. 2011;717:55–67.

201. Landry JP, Fei Y, Zhu XD. High throughput, label-free screen-

ing small molecule compound libraries for protein-ligands using

combination of small molecule microarrays and a special

ellipsometry-based optical scanner. Int Drug Discov. December

2011:8–13.

202. Landry JP, Fei Y, Zhu XD. Simultaneous measurement of

10,000 protein-ligand affinity constants using microarray-

based kinetic constant assays. Assay Drug Dev Technol. June

2012:250–259.

203. Larsson LI. Fixation and tissue pretreatment. In: Larsson L-I, ed.

Immunocytochemistry: Theory and Practice. Boca Raton, FL:

CRC Press; 1988:41–76.

204. Leach MW, Halpern WG, Johnson CW, et al. Use of tissue

cross-reactivity studies in the development of antibody-based

biopharmaceuticals: history, experience, methodology, and

future directions. Toxicol Pathol. 2010;38(7):1138–1166.

205. Leong AS-Y, Gilham PN. The effects of progressive formalde-

hyde fixation on the preservation of tissue antigens. Pathology.

1989;21(4):266–268.

206. Leong AS-Y, Leong TY-M. Newer developments in immuno-

histology. J Clin Pathol. 2006;59(11):1117–1126.

207. Leong AS-Y, Suthipintawong C, Vinyuvat S. Immunostaining of

cytologic preparations: a review of technical problems. Appl


208. Leong TY-M, Cooper K, Leong AS-Y. Immunohistology: past,

present, and future. Adv Anat Pathol. 2010;17(6):404–418.

209. Levenson R, Beechem J, McNamara G. Spectral imaging in pre-

clinical research and clinical pathology. Anal Cell Pathol. 2012;

35(5–6):339–361.

210. Lhotka JF, Ferreira AV. A comparison of deformalinizing tech-

nics. Stain Technol. 1949;25(1):27–32.

211. Liguori MJ, Hoff-Velk JA, Ostrow DH. Recombinant human

interleukin-6 enhances the immunoglobulin secretion of a

rabbit-rabbit hybridoma. Hybridoma. 2001;20(3):189–198.

212. Lin Y, Hatem J, Wang J, et al. Tissue microarray–based immuno-

histochemical study can significantly underestimate the expression

of HER2 and progesterone receptor in ductal carcinoma in situ of

the breast. Biotech Histochem. 2011;86(5):345–350.

213. Lipman NS, Jackson LR, Trudel LJ, et al. Monoclonal versus

polyclonal antibodies: distinguishing characteristics, applica-

tions, and information resources. Inst Lab Anim Res J. 2005;

46(3):258–268.

214. Liu J, Lau SK, Varma VA, et al. Multiplexed detection and char-

acterization of rare tumor cells in Hodgkin’s lymphoma with

multicolor quantum dots. Anal Chem. 2010;82(14):637–643.

215. Liu J, Lau SK, Varma VA, et al. Molecular mapping of tumor

heterogeneity on clinical tissue specimens with multiplexed

quantum dots. ACS Nano. 2010;4(5):2755–2765.

216. Lizardi PM, Huang X, Zhu Z, et al. Mutation detection and

single-molecule counting using isothermal rolling-circle ampli-

fication. Nat Genet. 1998;19(3):225–232.

217. Login GR, Beary ES, Brown HS, et al. Microwave Device Use in

the Histology Laboratory: Approved Guideline. CLSI document

GP28-A. Wayne, PA: Clinical and Laboratory Standards Insti-

tute; 2005.

218. Lucocq JM, Roth J. Colloidal gold and colloidal silver-metallic

markers for light microscopic histochemistry. In: Immunocyto-

chemistry. New York, NY: Academic Press; 1985:203–236.

219. Malmstrom PU, Wester K, Vasko J, et al. Expression of prolif-

erative cell nuclear antigen (PCNA) in urinary bladder carci-

noma: evaluation of antigen retrieval methods. Acta Pathol

Microbiol Immunol Scand. 1992;100(11):988–992.

220. Mandel U, Gaggero B, Reibel J, et al. Oncofetal fibronectins in

oral carcinomas: correlation of two different types. APMIS.

1994;102(9):695–702.

221. Mandel U, Therkildsen MH, Reibel J, et al. Cancer-associated

changes in glycosylation of fibronectin: immunohistological

localisation of oncofetal fibronectin defined by monoclonal anti-

bodies. APMIS. 1992;100(9):817–826.

222. Mandell JW. Phosphorylation state-specific antibodies: applica-

tions in investigative and diagnostic pathology. Am J Pathol.

2003;163(5):1687–1698.

223. Mandell JW. Immunohistochemical assessment of protein phos-

phorylation state: the dream and the reality. Histochem Cell Biol.

2008;130(3):465–471.

224. Mangham DC, Bradwell AR, Isaacson PG. MICA: a highly sen-

sitive and avidin-free immunohistochemical detection system.

Adv Anat Pathol. 2000;7(6):360–364.

225. Mangham DC, Isaacson PG. A novel immunohistochemical

detection system using mirror image complementary antibodies

(MICA). Histopathology. 1999;35(2):129–133.

226. Mansfield JR. A colorful path, present and future: a review of

multispectral imaging methods in anatomic pathology. Vet

Pathol. 2014;51(1). doi: 10.1177/0300985813506918

227. Martın CA, Salomoni PD, Badran AF. Cytokeratin immunoreac-

tivity in mouse tissues: study of different antibodies with a new

detection system. Appl Immunohistochem Mol Morphol. 2001;

9(1):70–73.

228. Martinet W, Schrijvers DM, Timmermans J-P, et al. Immunohis-

tochemical analysis of macroautophagy: recommendations and

limitations. Autophagy. 2013;9(3):1–17.

229. Mason DY, Erber WN, Falini B, et al. Immunoenzymatic label-

ing of hematological samples with monoclonal antibodies. In:

Beverly PCL, ed. Monoclonal Antibodies. New York, NY:

Churchill Livingstone; 1986:145–181.

230. Mason DY, Sammons R. Alkaline phosphatase and peroxidase

for double immunoenzymatic labelling of cellular constituents.

J Clin Pathol. 1978;31(5):454–460.

231. Mason JT, O’Leary TJ. Effects of formaldehyde fixation on protein

secondary structure: a calorimetric and infrared spectroscopic

investigation. J Histochem Cytochem. 1991;39(2):225–239.

232. Mathews JB, Mason GI. Influence of decalcifying agents on

immunoreactivity of formalin-fixed, paraffin-embedded tissue.

Histochem J. 1984;16(7):771–787.

233. McNeilly F, Kennedy S, Moffett D, et al. A comparison of in situ

hybridization and immunohistochemistry for the detection of a

new porcine circovirus in formalin-fixed tissues from pigs with

post-weaning multisystemic wasting syndrome (PMWS). J Virol

Methods. 1999;80(2):123–128.




234. Merz H, Malisius R, Mann-Weiler S, et al. ImmunoMax: a max-

imized immunohistochemical method for the retrieval and

enhancement of hidden antigens. Lab Invest. 1995;73(1):

149–156.

235. Metz B, Kersten GFA, Hoogerhout P, et al. Identification of

formaldehyde-induced modifications in proteins: reactions with

model peptides. J Biol Chem. 2004;279(8):6235–6243.

236. Miettinen M. Immunostaining of intermediate filament proteins

in paraffin sections: evaluation of optimal protease treatment to

improve the immunoreactivity. Path Res Pract. 1989;184(4):

431–436.

237. Mighell AJ, Hume WJ, Robinson PA. An overview of the com-

plexities and subtleties of immunohistochemistry. Oral Dis.

1998;4(3):217–223.

238. Miller MA, Ramos-Vara JA, Kleiboeker SB, et al. Effects of

delayed or prolonged fixation on immunohistochemical detec-

tion of bovine viral diarrhea virus type I in skin of two persis-

tently infected calves. J Vet Diagn Invest. 2005;17(5):461–463.

239. Miller RT. What every pathologist needs to know about techni-

cal immunohistochemistry (or how to avoid ‘‘immune confu-

sion’’). Paper presented at: American Academy of Oral

Maxillofacial Pathology Annual Meeting; April 30, 2011; San

Juan, Puerto Rico.

240. Miller RT, Kubier P. Immunohistochemistry on cytologic speci-

mens and previously stained slides (when no paraffin block is

available). J Histotechnol. 2002;25(4):251–257.

241. Mizogami M, Sakurai S, Kikuchi M, et al. Application of the

AMeX method to the evaluation of HER-2 status in breast carci-

nomas: comparison with results of HercepTest. Pathol Internat.

2003;53(1):27–29.

242. Moatamed NA, Nanjangud G, Pucci R, et al. Effect of ischemic

time, fixation time, and fixative type on HER2/neu immunohis-

tochemical and fluorescence in situ hybridization results in

breast cancer. Am J Clin Pathol. 2011;136(5):754–761.

243. Mobasheri A, Airley R, Foster CS, et al. Post-genomic applica-

tions of tissue microarrays: basic research, prognostic oncology,

clinical genomics and drug discovery. Histol Histopathol. 2004;

19(1):325–335.

244. Moelans CB, ter Hoeve N, van Ginkel J-W, et al. Formaldehyde

substitute fixatives: analysis of macroscopy, morphologic analy-

sis, and immunohistochemical analysis. Am J Clin Pathol. 2011;

136(4):548–556.

245. Montero C. The antigen-antibody reaction in immunohisto-

chemistry. J Histochem Cytochem. 2003;51(1):1–4.

246. Moore PF, Rodriguez-Bertos A, Kass PH. Feline gastrointestinal

lymphoma: mucosal architecture, immunophenotype, and mole-

cular clonality. Vet Pathol. 2012;49(4):658–668.

247. Morgan JM, Navabi H, Jasani B. Role of calcium chelation in

high-temperature antigen retrieval at different pH values. J

Pathol. 1997;182(2):233–237.

248. Morgan JM, Navabi H, Schmid KW, et al. Possible role of

tissue-bound calcium ions in citrate-mediated high-temperature

antigen retrieval. J Pathol. 1994;174(4):301–307.

249. Moriguchi K, Mitamura Y, Iwami J, et al. Energy filtering trans-

mission electron microscopy immunocytochemistry and antigen

retrieval of surface layer proteins from Tannerella forsythensis

using microwave or autoclave heating with citraconic anhydride.

Biotech Histotech. 2012;87(8):485–493.

250. Mount SL, Cooper K. Beware of biotin: a source of false-

positive immunohistochemistry. Cur Diagn Pathol. 2001;7(3):

161–167.

251. Myers J. A review of automated slide stainers for IHC and ISH.

Med Lab Obs. January 2008:41–44.

252. Naito Y, Okabe Y, Kawahara A, et al. Guide to diagnosing pri-

mary pancreatic lymphoma, B-cell type: immunohistochemistry

improves the diagnostic accuracy of endoscopic

ultrasonography-guided fine needle aspiration cytology. Diagn

Cytopathol. 2011;40(8):732–736.

253. Namimatsu S, Ghazizadeh M, Sugisaki Y. Reversing the effects

of formalin fixation with citraconic anhydride and heat: a univer-

sal antigen retrieval method. J Histochem Cytochem. 2005;

53(1):3–11.

254. Nelson PN, Fletcher SM, MacDonald D, et al. Assay restriction

profiles of three monoclonal antibodies recognizing the G3m(u)

allotype: development of an allotype specific assay. J Immunol

Methods. 1991;138(1):57–64.

255. Nelson PN, Reynolds GM, Waldron EE, et al. Demystified . . .

monoclonal antibodies. J Clin Pathol Mol Pathol. 2000;53(3):

111–117.

256. Nielsen TO, Hsu FD, O’Connell JX, et al. Tissue microarray

validation of epidermal growth factor receptor and SALL2 in

synovial sarcoma with comparison to tumors of similar histol-

ogy. Am J Pathol. 2003;163(4):1449–1456.

257. Nikiel B, Chekan M, Jarzab M, et al. Endogenous avidin biotin

(EABA) in thyroid pathology: immunohistochemical study.

Thyroid Res. 2009;2:5.

258. Nishizuka S, Chen S-T, Gwadry FG, et al. Diagnostic markers

that distinguish colon and ovarian adenocarcinomas: identifica-

tion by genomic, proteomic, and tissue array profiling. Cancer

Res. 2003;63(17):5243–5250.

259. Okada H, Iwamaru Y, Fukuda S, et al. Detection of disease-

associated prion protein in the optic nerve and the adrenal gland

of cattle with bovine spongiform encephalopathy by using

highly sensitive immunolabeling procedures. J Histochem

Cytochem. 2012;60(4):290–300.

260. Olapade-Olaopa E, Ogunbiyi JO, MacKay EH, et al. Further

characterization of storage-related alterations in immunoreactiv-

ity of archival tissue sections and its implications for collabora-

tive multicenter immunohistochemical studies. Appl


261. O’Leary TJ, Fowler CB, Evers DL, et al. Protein fixation and

antigen retrieval: chemical studies. Biotech Histochem. 2009;

84(5):217–221.

262. Olszewski WL, Zolich D, Manokaran G, et al. Sodium chloride

fixation of tissues under field conditions in tropical countries. J

Immunol Methods. 2004;284(1–2):39–44.

263. Ordonez NG, Manning JT, Brooks TE. Effect of trypsinization

on the immunostaining of formalin-fixed, paraffin-embedded

tissues. Am J Surg Pathol. 1988;12(2):121–129.

264. Otali D, Stockard CR, Oelschlager DK, et al. Combined effects

of formalin fixation and tissue processing on immunorecogni-

tion. Biotech Histochem. 2009;84(5):223–247.




265. Packeisen J, Buerger H, Krech R, et al. Tissue microarrays: a

new approach for quality control in immunohistochemistry. J

Clin Pathol. 2002;55(8):613–615.

266. Packeisen J, Korsching E, Herbst H, et al. Demystified . . . tissue

microarray technology. J Clin Pathol. 2003;56(4):198–204.

267. Pang Y, von Turkovich M, Wu H, et al. The binding of thyroid

transcription factor-1 and hepatocyte paraffin 1 to mitochondrial

proteins in hepatocytes: a molecular and immunoelectron micro-

scopic study. Am J Clin Pathol. 2006;125(5):722–726.

268. Pericleus P, Gazouli M, Lyberopoulou A, et al. Quantum dots

hold promise for early cancer imaging and detection. Int J

Cancer. 2012;131(3):519–1528.

269. Petrosyan K, Tamayo R, Joseph D. Sensitivity of a novel

biotin-free detection reagent (Powervisionþ™) for immunohis-

tochemistry. J Histotechnol. 2002;25(4):247–250.

270. Petryaueva E, Algar WR, Medintz IL. Quantum dots in bioanalysis:

a review of applications across various platforms for fluorescence

spectroscopy and imaging. Appl Spectr. 2013;67(3):215–252.

271. Pileri SA, Roncador G, Ceccarelli C, et al. Antigen retrieval

techniques in immunohistochemistry: comparison of different

methods. J Pathol. 1997;183(1):116–123.

272. Pinhel IF, MacNeill FA, Hills MJ, et al. Extreme loss of immu-

noreactive p-Akt and p-Erk1/2 during routine fixation of primary

breast cancer. Breast Cancer Res. 2010;12(5):R76.

273. Pinkus GS, O’Connor EM, Etheridge CL, et al. Optimal immunor-

eactivity of keratin proteins in formalin-fixed, paraffin-embedded

tissue requires preliminary trypsinization: an immunoperoxidase

study of various tumors using polyclonal and monoclonal antibo-

dies. J Histochem Cytochem. 1985;33(5):465–473.

274. Pirici D, Mogoanta L, Kumar-Singh S, et al. Antibody elution

method for multiple immunohistochemistry on primary

antibodies raised in the same species and of the same subtype.

J Histochem Cytochem. 2009;57(6):567–575.

275. Polak JM, Van Noorden S. Introduction to Immunocytochemis-

try. 3rd ed. Oxford, UK: Bios Scientific Publishers Ltd; 2003.

276. Ponten F, Jirstrom K, Uhlen M. The human protein atlas: a tool

for pathology. J Pathol. 2008;216(4):387–393.

277. Pool CW, Buijs RM, Swaab DF, et al. On the way to a specific

immunocytochemical localization. In: Cuello AC, ed. Neuroim-

muno-cytochemistry. IBRO Handbook Series: Methods in

Neurosciences. Chichester, England: John Wiley; 1983:1–46.

278. Popkov M, Mage RG, Alexander CB, et al. Rabbit immune

repertoires as sources for therapeutic monoclonal antibodies: the

impact of kappa allotype-correlated variation in cysteine content

on antibody libraries selected by phage display. J Mol Biol.

2003;325(2):325–335.

279. Pradidarcheep W, Labruyere WT, Dabhoiwala NF, et al. Lack of

specificity of commercially available antisera: better specifica-

tions needed. J Histochem Cytochem. 2008;56(12):1099–1111.

280. Prento P, Lyon H. Commercial formalin substitutes for histo-

pathology. Biotech Histotech. 1997;72(5):273–282.

281. Prioleau J, Schnitt SJ. p53 antigen loss in stored paraffin slides.

N Engl J Med. 1995;332(22):1521–1522.

282. Putchler H, Meloan SN. On the chemistry of formaldehyde

fixation and its effects on immunohistochemical reactions.

Histochemistry. 1985;82(3):201–204.

283. Rader C. Antibody libraries in drug and target discovery. Drug

Discov Today. 2001;6(1):36–43.

284. Ramos-Vara JA. Technical aspects of immunohistochemistry.

Vet Pathol. 2005;42(4):405–426.

285. Ramos-Vara JA. Immunohistochemical methods. In: Howard

GC, Kaser MR, eds. Making and Using Antibodies: A Practical

Handbook. 2nd ed. Boca Raton, FL: CRC Press; 2013:303–341.

286. Ramos-Vara JA, Averie A, Averie P. Advanced diagnostic

techniques. In: Raskin R, Meyer D, eds. Canine and Feline

Cytology: A Color Atlas and Interpretation Guide. 2nd ed. St

Louis, MO: Elsevier; 2010:395–437.

287. Ramos-Vara JA, Beissenherz M. Optimization of immunohisto-

chemical methods using two different antigen retrieval methods

on formalin-fixed, paraffin-embedded tissues: experience with

63 markers. J Vet Diagn Invest. 2000;12(4):307–311.

288. Ramos-Vara JA, Beissenherz ME, Miller MA, et al. Retrospec-

tive study of 338 canine oral melanomas with clinical, histolo-

gic, and immunohistochemical review of 129 cases. Vet

Pathol. 2000;37(6):597–608.

289. Ramos-Vara JA, Beissenherz ME, Miller MA, et al. Immunor-

eactivity of A103, an antibody to Melan A, in canine steroid-

producing tissues and their tumors. J Vet Diagn Invest. 2001;

13(4):328–332.

290. Ramos-Vara JA, Frank CB, DuSold D, et al. Immunohistochem-

ical expression of melanocytic antigen PNL2, Melan A, S100

and PGP 9.5 in equine melanocytic neoplasms [published online

January 31, 2013]. Vet Pathol. doi:10.1177/0300985812471545.

291. Ramos-Vara JA, Kiupel M, Baszler T, et al. Suggested

guidelines for immunohistochemical techniques in veterinary

diagnostic laboratories. J Vet Diagn Invest. 2008;20(4):393–413.

292. Ramos-Vara JA, Kiupel M, Miller MA. Veterinary diagnostic

immunohistochemistry: a survey of 47 laboratories. J Histotech-

nol. 2005;28(1):19–23.

293. Ramos-Vara JA, Miller MA. Comparison of two polymer-based

immunohistochemical detection systems: ENVISIONþ™ and

ImmPRESS™. J Microsc. 2006;224(2):135–139.

294. Ramos-Vara JA, Miller MA. Immunohistochemical detection of

protein gene product 9.5 (PGP 9.5) in canine epitheliotropic T-

cell lymphoma (mycosis fungoides). Vet Pathol. 2007;44(1):74–79.

295. Ramos-Vara JA, Miller MA. Immunohistochemical identifica-

tion of canine melanocytic neoplasms with antibodies to

melanocytic antigen PNL2 and tyrosinase: comparison with

Melan A. Vet Pathol. 2011;48:443–450.

296. Ramos-Vara JA, Miller MA. Immunohistochemical expression

of E-cadherin does not distinguish canine cutaneous histiocy-

toma from other canine round cell tumors. Vet Pathol. 2011;

48(3):758–763.

297. Ramos-Vara JA, Miller MA, Boucher M, et al. Immunohisto-

chemical detection of uroplakin III, cytokeratin 7, and

cytokeratin 20 in canine urothelial tumors. Vet Pathol. 2003;

40(1):55–62.

298. Ramos-Vara JA, Miller MA, Johnson GC. Immunohistochem-

ical characterization of canine hyperplastic hepatic lesions and

hepatocellular and biliary neoplasms with monoclonal antibody

hepatocyte paraffin 1 and a monoclonal antibody to cytokeratin

7. Vet Pathol. 2001;38(6):636–643.




299. Ramos-Vara JA, Miller MA, Johnson GC. Usefulness of thyroid

transcription factor-1 immunohistochemical staining in the

differential diagnosis of primary pulmonary tumors of dogs. Vet

Pathol. 2005;42(3):315–320.

300. Ramos-Vara JA, Miller MA, Johnson GC, et al. Immunohisto-

chemical detection of thyroid transcription factor-1, thyroglobu-

lin, and calcitonin in canine normal, hyperplastic, and neoplastic

thyroid gland. Vet Pathol. 2002;39(4):480–487.

301. Ramos-Vara JA, Miller MA, Johnson GC, et al. Melan A and

S100 protein immunohistochemistry in feline melanomas: 48

cases. Vet Pathol. 2002;39(1):127–132.

302. Ramos-Vara JA, Miller MA, Valli VEO. Immunohistochemical

detection of multiple myeloma 1/interferon regulatory factor 4

(MUM1/IRF-4) in canine plasmacytoma: comparison with

CD79a and CD20. Vet Pathol. 2007;44(6):875–884.

303. Ramos-Vara JA, Saeteele J. Immunohistochemistry. In: Howard

GC, Kaser MR, eds. Making and Using Antibodies: A Practical

Handbook. Boca Raton, FL: CRC Press; 2007:273–314.

304. Ramos-Vara JA, Webster JD, DuSold D, et al. Immunohisto-

chemical evaluation of the effects of paraffin section storage

on biomarker stability [published online February 22, 2013]. Vet

Pathol. doi:10.1177/0300985813476067.

305. Rangel CS. The tissue microarray: helpful hints! J Histotechnol.

2002;25(2):93–100.

306. Rasmussen OF. Controls. In: Key M, ed. Immunohistochemical

Staining Methods. 4th ed. Carpinteria, CA: Dako Corporation;

2006:113–118.

307. Reid V, Doherty J, McIntosh G, et al. The first quantitative com-

parison of immunohistochemical rabbit and mouse monoclonal

antibody affinities using Biacore analysis. J Histotechnol.

2007;30(3):177–182.

308. Reiner-Concin A. External quality assurance in immunohisto-

chemistry: is it the solution to a complex problem? Breast Care

2008;3(2):78–79.

309. Rhodes A, Jasani B, Barnes DM, et al. Reliability of immunohisto-

chemical demonstration of estrogen receptors in routine practice:

interlaboratory variance in the sensitivity of detection and evalua-

tion of scoring systems. J Clin Pathol. 2000;53(2):125–130.

310. Rhodes A, Jasani B, Couturier J, et al. A formalin-fixed,

paraffin-processed cell line standard for quality control of

immunohistochemical assay of HER-2/new expression in breast

cancer. Am J Clin Pathol. 2002;117(1):81–89.

311. Rizzardi A, Johnson AT, Vogel RI, et al. Quantitative comparison

of immunohistochemical staining measured by digital image anal-

ysis versus pathologist visual scoring. Diagn Pathol. 2012;7:42.

312. Roh MH, Schmidt L, Placido J, et al. The application and diag-

nostic utility of immunocytochemistry on direct smears in the

diagnosis of pulmonary adenocarcinoma and squamous cell car-

cinoma. Diagn Cytopathol. 2012;40(11):949–955.

313. Rossi S, Laurino L, Furlanetto A, et al. A comparative study

between a novel category of immunoreagents and the corre-

sponding mouse monoclonal antibodies. Am J Clin Pathol.

2005;124(2):295–302.

314. Rudiger T, Hofler H, Kreipe HH, et al. Quality assurance in

immunohistochemistry: results of interlaboratory trial involving

172 pathologists. Am J Surg Pathol. 2002;26(7):873–882.

315. Russo D, Ambrosino A, Vittoria A, et al. Signal amplification by

combining two advanced immunohistochemical techniques. Eur

J Histochem. 2003;47(4):379–384.

316. Russo G, Zegar C, Giordano A. Advantages and limitations of

microarray technology in human cancer. Oncogene. 2003;

22(42):6497–6507.

317. Sabatini DD, Bensch K, Barrnett RJ. Cytochemistry and electron

microscopy: the preservation of cellular ultrastructure and enzy-

matic activity by aldehydes fixation. J Cell Biol. 1963;17(1):

19–58.

318. Sabattini E, Bisgaard K, Ascani S, et al. The EnVision™þsystem: a new immunohistochemical method for diagnostics and

research. Critical comparison with the APAAP, ChemMate™,

CSA, LABC, and SABC techniques. J Clin Pathol. 1998;

51(4):506–511.

319. Sabattini S, Bettini G. Prognostic value of histologic and immu-

nohistochemical features in feline cutaneous mast cell tumor.

Vet Pathol. 2010;47(4):643–653.

320. Saper CB. A guide to the perplexed on the specificity of antibo-


321. Saper CB, Sawchenko PE. Magic peptides, magic antibodies:

guidelines for appropriate controls for immunohistochemistry.

J Comp Neurol. 2003;465(2):161–163.

322. Sato Y, Mukai K, Watanabe S, et al. The AMeX method: a sim-

plified technique of tissue processing and paraffin embedding

with improved preservation of antigens for immunostaining.

Am J Pathol. 1986;125(3):431–435.

323. Savage EC, DeYoung BR. Antibody expiration in the context of

resource limitation: what is the evidence basis? Am J Clin

Pathol. 2010;134(1):60–64.

324. Saxena R, Badve S. Tissue microarray: construction and quality

assurance. In: Kumar GL, Rudbeck L, eds. Immunohistochem-

ical Staining Methods. 5th ed. Carpinteria, CA: Dako; 2009:

43–50.

325. Schweitzer B, Wiltshire S, Lambert J, et al. Immunoassays with

rolling circle DNA amplification: a versatile platform for ultra-

sensitive antigen detection. Proc Natl Acad Sci U S A. 2000;

97(18):10113–10119.

326. Seidal T, Balaton AJ, Battifora H. Interpretation and quantifica-

tion of immunostains. Am J Surg Pathol. 2001;25(9):1204–1207.

327. Seidu MA, Adams AR, Gyasi RK, et al. Immunoreactivity of

some epitopes in longtime inappropriately stored paraffin-

embedded tissues. J Histotechnol. 2013;36(2):59–64.

328. Sharp PA. Split genes and RNA splicing. Cell. 1994;77(6):

805–815.

329. Sherman ME, Jimenez-Joseph D, Gangi MD, et al. Immunos-

taining of small cytologic specimens: facilitation with cell

transfer. Acta Cytol. 1994;38(1):18–22.

330. Shi S-R, Cote RJ, Taylor CR. Antigen retrieval immunohisto-

chemistry: past, present, and future. J Histochem Cytochem.

1997;45(3):327–343.

331. Shi S-R, Cote RJ, Taylor CR. Standardization and further

development of antigen retrieval immunohistochemistry: strate-

gies and future goals. J Histotechnol. 1999;22(3):177–192.

332. Shi S-R, Cote RJ, Taylor CR. Antigen retrieval techniques: cur-

rent perspectives. J Histochem Cytochem. 2001;49(8):931–937.




333. Shi S-R, Cote RJ, Young LL, et al. Antigen retrieval immunohis-

tochemistry: practice and development. J Histotechnol. 1997;

20(2):145–154.

334. Shi S-R, Gu J, Turrens J, et al. Development of the antigen retrie-

val technique: philosophical and theoretical bases. In: Shi S-R,

Gu J, Taylor CR, eds. Antigen Retrieval Techniques: Immuno-

histochemistry and Molecular Morphology. Natick, MA: Eaton

Publishing; 2000:17–40.

335. Shi S-R, Guo J, Cote RJ, et al. Sensitivity and detection

efficiency of a novel two-step detection system (PowerVision)

for immunohistochemistry. Appl Immunohistochem Mol

Morphol. 1999;7(3):201–208.

336. Shi S-R, Imam S, Young L, et al. Antigen retrieval immunohis-

tochemistry under the influence of pH using monoclonal antibo-


337. Shi S-R, Key ME, Kalra KL. Antigen retrieval in formalin-fixed,

paraffin-embedded tissue: an enhancement method for immuno-

histochemical staining based on microwave oven heating of

tissue sections. J Histochem Cytochem. 1991;39(6):741–748.

338. Shi S-R, Liu C, Pootrakul L, et al. Evaluation of the value of

frozen tissue section used as ‘‘gold standard’’ for immunohisto-

chemistry. Am J Clin Pathol. 2008;129(3):358–366.

339. Shi S-R, Shi Y, Taylor CR. Antigen retrieval immunohistochem-

istry: a review and future prospects in research and diagnosis

over two decades. J Histochem Cytochem. 2011;59(1):13–32.

340. Shi S, Cheng Q, Zhang P, et al. Immunofluorescence with dual

microwave retrieval of paraffin-embedded sections in the assess-

ment of human renal biopsy specimens. Am J Clin Pathol. 2013;

139(1):71–78.

341. Shtilbans V, Szporn AH, Wu M, et al. p63 immunostaining in

destained bronchoscopic cytological specimens. Diagn Cyto-

pathol. 2005;32(4):198–203.

342. Sickel JZ, di Sant’Agnese PA. Anomalous immunostaining of

‘‘optically clear’’ nuclei in gestational endometrium. Arch

Pathol Lab Med. 1994;118(8):831–833.

343. Siddiqui S, Rimm DL. Pre-analytical variables and phospho-

specific antibodies: the Achilles heel of immunohistochemistry.

Breast Cancer Res. 2010;12(6):113.

344. Simon R, Mirlacher M. Tissue microarrays for translational

research. In: Jordan B, ed. Microarrays in Diagnostics and Bio-

marker Development. Berlin, Germany: Springer-Verlag; 2012:

135–152.

345. Skoog L, Tani E: Immunocytochemistry: an indispensable tech-

nique in routine cytology. Cytopathology. 2011;22(4):215–229.

346. Skotheim RI, Abeler VM, Nesland JM, et al. Candidate genes for

testicular cancer evaluated by in situ protein expression analysis

in tissue microarrays. Neoplasia. 2003;5(5):397–404.

347. Sompuram SR, Vani K, Bogen SA. A molecular model of

antigen retrieval using a peptide array. Am J Clin Pathol.

2006;125(1):91–98.

348. Sompuram SR, Vani K, Hafer LJ, et al. Antibodies immunoreac-

tive with formalin-fixed tissue antigens recognize linear protein

epitopes. Am J Clin Pathol. 2006;125(1):82–90.

349. Sompuram SR, Vani K, Messana E, et al. A molecular mechan-

ism of formalin fixation and antigen retrieval. Am J Clin Pathol.

2004;121(2):190–199.

350. Speel EJM, Hopman AHN, Komminoth P. Amplification meth-

ods to increase the sensitivity of in situ hybridization: play

CARD(S). J Histochem Cytochem. 1999;47(3):281–288.

351. Spicer SS, Spivey MA, Ito M, et al. Some ascites monoclonal

antibody preparations contain contaminants that bind to selected

Golgi zones or mast cells. J Histochem Cytochem. 1994;42(2):

213–221.

352. Srinivasan M, Sedmark D, Jewell S. Effect of fixatives and tissue

processing on the content and integrity of nucleic acids. Am J

Pathol. 2002;161(6):1961–1971.

353. Srinivasappa J, Saegusa J, Prabhakar BS, et al. Molecular mimi-

cry: frequency of reactivity of monoclonal antiviral antibodies

with normal tissues. J Virol. 1986;57(1):397–401.

354. Stanta G. Archive tissues. In: Stanta G, ed. Guidelines for Mole-

cular Analysis in Archived Tissues. Berlin, Germany: Springer-

Verlag; 2011:3–4.

355. Stanta G, Carcangiu ML, Rosai J. The biochemical and immuno-

histochemical profile of thyroid neoplasia. Pathol Annu. 1988;

23(1):129–157.

356. Start RD, Flynn MS, Cross SS, et al. Is the grading of breast car-

cinomas affected by a delay in fixation? Virch Arch A Pathol

Anat. 1991;419(6):475–477.

357. Sternberger LA, Hardy PH, Cuculis JJ, et al. The unlabeled anti-

body enzyme method of immunohistochemistry: preparation and

properties of soluble antigen-antibody complex (horseradish

peroxidase–antihorseradish peroxidase) and its use in identifica-

tion of spirochetes. J Histochem Cytochem. 1970;18(5):

315–333.

358. Storz M, Moch H. Tissue microarrays and biomarker validation

in molecular diagnostics. In: Cheng L, Zhang DY, Eble JN, eds.

Molecular Genetic Pathology. New York, NY: Springer; 2013:

455–463.

359. Stumm MM, Walker MR, Stork C, et al. Validation of a post-

fixation tissue storage and transport medium to preserve histo-

pathology and molecular pathology analyses (total and

phosphoactivated proteins, and FISH). Am J Clin Pathol.

2012;137(3):429–436.

360. Sullivan CA, Chung GG. Biomarker validation: in situ analysis of

protein expression using semiquantitative immunohistochemistry-

based techniques. Clin Colorectal Cancer. 2008;7(3):172–177.

361. Suthipintawong C, Leong ASY, Chan K-W, et al. Immunostaining

of estrogen receptor, progesterone receptor, MIb1 antigen, and c-

erbB-2 oncoprotein in cytologic specimens: a simplified method

with formalin fixation. Diagn Cytopathol. 1997;17(2):127–133.

362. Suthipintawong C, Leong ASY, Vinyuvat S. Immunostaining of

cell preparations: a comparative evaluation of common fixatives

and protocols. Diagn Cytopathol. 1996;15(2):167–174.

363. Suzuki Y, Imada T, Yamaguchi I, et al. Effects of prolonged

water washing of tissue samples fixed in formalin on histological

staining. Biotech Histochem. 2012;87(4):241–248.

364. Svistunova DM, Musinova YR, Polyakov VY, et al. A simple

method for the immunocytochemical detection of proteins inside

nuclear structures that are inaccessible to specific antibodies. J


365. Swaab DF. Comments on the validity of immunocytochemical

methods. In: Chan-Palay V, Palay SL, eds. Neurology and




Neurobiology: Cytochemical Methods in Neuroanatomy. Vol. 1.

New York, NY: Alan R. Liss; 1982:423–440.

366. Syrbu SI, Cohen MB. An enhanced antigen-retrieval protocol for

immunohistochemical staining of formalin-fixed, paraffin-

embedded tissues. In: Kalyuzhny AE, ed. Signal Transduction

Immunohistochemistry: Methods and Protocols. New York,

NY: Humana; 2011:101–110.

367. Takahashi M, Yang XJ, Sugimura J, et al. Molecular subclassi-

fication of kidney tumors and the discovery of new diagnostic

markers. Oncogene. 2003;22(43):6810–6818.

368. Taylor CR. Report of the Immunohistochemistry Steering

Committee of the Biological Stain Commission. Proposed for-

mat: package insert for immunohistochemistry products. Biotech

Histochem. 1992;67(6):323–338.

369. Taylor CR. Quantifiable internal reference standards for immu-

nohistochemistry: the measurement of quantity by weight. Appl


370. Taylor CR, Shi S-R, Barr NJ, et al. Techniques of immunohisto-

chemistry: principles, pitfalls, and standardization. In: Dabbs DJ,

ed. Diagnostic Immunohistochemistry. New York, NY: Church-

ill Livingstone; 2002:3–43.

371. Taylor CR, Shi S-R, Barr NJ, et al. Techniques of immunohisto-

chemistry: principles, pitfalls, and standardization. In: Dabbs DJ,

ed. Diagnostic Immunohistochemistry: Theranostic and Genomic

Applications. 3rd ed. Philadelphia, PA: Saunders; 2010:1–41.

372. Teruya-Feldstein J. The immunohistochemistry laboratory:

looking at molecules and preparing for tomorrow. Arch Pathol

Lab Med. 2010;134(11):1659–1665.

373. Thompson JJ, Yager JA, Best SJ, et al. Canine subcutaneous

mast cell tumors: cellular proliferation and kit expression as

prognostic markers. Vet Pathol. 2011;48(1):169–181.

374. Thrustfield M. Veterinary Epidemiology. 2nd ed. Oxford, UK:

Blackwell Science; 1995.

375. Torlakovic EE, Riddell R, Banerjee D, et al. Canadian Associa-

tion of Pathologists National Standards Committee/Immunohis-

tochemistry. Am J Clin Pathol. 2010;133(3):354–365.

376. True LD. Quality control in molecular immunohistochemistry.

Histochem Cell Biol. 2008;130(3):473–480.

377. Tubbs RR, Nagle R, Leslie K, et al. Extension of useful reagent

shelf life beyond manufacturer’s recommendations. Arch Pathol

Lab Med. 1998;122(12):1051–1052.

378. Uhlen M, Oksvold P, Fagerberg L, et al. Towards a knowledge-

based human protein atlas. Nat Biotech. 2010;28(12):1248–1250.

379. Valli V, Peters E, Williams C, et al. Optimizing methods in

immunocytochemistry: one laboratory’s experience. Vet Clin

Pathol. 2009;38(2):261–269.

380. van Bogaert L-J. p16 INK4a immunocytochemistry/immunohisto-

chemistry: need for scoring uniformization to be clinically useful

in gynecological pathology. Ann Diagn Pathol. 2012;16(5):

422–426.

381. van de Rijn M, Gilks CB. Applications of microarrays to

histopathology. Histopathology. 2004;44(2):97–108.

382. van der Loos CM. Immunoenzyme Multiple Staining Method-

sods. New York, NY: Bios Scientific Publishers Ltd; 1999.

383. van der Loos CM. A focus on fixation. Biotech Histochem. 2007;

82(3):141–154.

384. van der Loos CM. Multiple immunoenzyme staining: methods

and visualizations for the observation with spectral imaging. J


385. van der Loos CM. Chromogens in multiple immunohistochem-

ical staining used for visual assessment and spectral imaging: the

colorful future. J Histotechnol. 2010;33(1):31–40.

386. van der Loos CM, Meijer-Jorna LB, Broekmans MEC, et al.

Anti-human vascular endothelial growth factor (VEGF) anti-

body selection for immunohistochemical staining of proliferat-

ing blood vessels. J Histochem Cytochem. 2010;58(2):109–118.

387. van der Loos CM, Teeling P. A generally applicable sequential

alkaline phosphatase immunohistochemical double staining. J

Histotechnol. 2008;31(3):119–127.

388. van Everbroeck B, Pals P, Martin J-J, et al. Antigen retrieval in

prion protein immunohistochemistry. J Histochem Cytochem.

1999;47(11):1465–1470.

389. van Gijlswijk RPM, Zijlmans HJMAA, Wiegant J, et al.

Fluorochrome-labeled tyramines: use in immunocytochemistry

and fluorescence in situ hybridization. J Histochem Cytochem.

1997;45(3):375–382.

390. van Hecke D. Routine immunohistochemical staining today:

choices to make, challenges to take. J Histotechnol. 2002;

25(1):45–54.

391. van Oss CJ, Absolom DR. Nature and thermodynamics of

antigen-antibody interactions. In: Atassi MZ, van Oss CJ, Abso-

lom DR, eds. Molecular Immunology. New York, NY: Marcel

Dekker; 1984:337–360.

392. van Sprundel RGHM, van den Ingh TSGAM, Desmet VJ, et al.

Keratin 19 marks poor differentiation and a more aggressive

behavior in canine and human hepatocellular tumors. Comp

Hepatol. 2010;9:4.

393. Varma M, Linden MD, Amin MB. Effect of formalin fixation

and epitope retrieval techniques on antibody 34bE12 immunos-

taining of prostatic tissues. Mod Pathol. 1999;12(5):472–478.

394. Varsegi GM, Shidham V. Cell block preparation from cytology

specimen with predominance of individually scattered cells. J

Visual Exper. 2009;29:e1316.

395. Vascellari M, Giantin M, Capello K, et al. Expression of Ki67,

BCL-2, and COX-2 in canine cutaneous mast cell tumors: associ-

ation with grading and prognosis. Vet Pathol. 2013;50(1):110–121.

396. Vilches-Moure JG, Ramos-Vara JA. Comparison of rabbit

monoclonal and mouse monoclonal antibodies in immunohisto-

chemistry in canine tissues. J Vet Diagn Invest. 2005;17(4):

346–350.

397. Vis AN, Kranse R, Nigg AL, et al. Quantitative analysis of the

decay of immunoreactivity in stored prostate needle biopsy sec-

tions. Am J Clin Pathol. 2000;113(3):369–373.

398. Vyberg M, Nielsen S. Dextran polymer conjugate two-step

visualization system for immunohistochemistry: a comparison

of EnVisionþ with two three-step avidin-biotin techniques. Appl

Immunohistochem. 1998;6(1):3–10.

399. Wakayabashi K, Sakata Y, Aoki NL. Conformation-specific

monoclonal antibodies to the calcium-induced structure of

protein C. J Biol Chem. 1986;261(24):11097–11105.

400. Ward JM. Controls in immunohistochemistry: is ‘‘brown’’ good

enough? Tox Pathol. 2004;32(3):273–274.




401. Warmington AR, Wilkinson JM, Riley CB. Evaluation of

ethanol-based fixatives as a substitute for formalin in diagnostic

clinical laboratories. J Histotechnol. 2000;23(4):299–308.

402. Webster JD, Dennis MM, Dervisis N, et al. Recommended

guidelines for the conduct and evaluation of prognostic studies

in veterinary oncology. Vet Pathol. 2011;48(1):7–18.

403. Webster JD, Miller MA, DuSold D, et al. Effects of prolonged

formalin fixation on diagnostic immunohistochemistry in

domestic animals. J Histochem Cytochem. 2009;57(8):753–761.

404. Webster JD, Miller MA, DuSold D, et al. Effects of prolonged

formalin fixation on the immunohistochemical detection of

infectious agents in formalin-fixed, paraffin-embedded tissues.

Vet Pathol. 2010;47(3):529–535.

405. Werner M, Chott A, Fabiano A, et al. Effect of formalin tissue

fixation and processing on immunohistochemistry. Am J Surg

Pathol. 2000;24(7):1016–1019.

406. Wester K, Wahlund E, Sundstrom C, et al. Paraffin section stor-

age and immunohistochemistry: effects of time, temperature,

fixation, and retrieval protocol with emphasis on p53 protein and

MIB1 antigen. Appl Immunohistochem Mol Morphol. 2000;8(1):

61–70.

407. Wetherington RW, Cooper HS, Al-Saleem T, et al. Clinical

significance of performing immunohistochemistry on cases with

a previous diagnosis of cancer coming to a national comprehen-

sive cancer center for treatment or second opinion. Am J Surg

Pathol. 2002;26(9):1222–1230.

408. Wick MR, Hagen KA, Frizzera G. Three immunostaining

techniques for the localization of leucocyte common antigen in

formalin fixed, paraffin embedded dermatological biopsy speci-

mens. Am J Dermatol. 1987;9(3):250–255.

409. Wick MR, Mills SE. Consensual interpretive guidelines for diag-

nostic immunohistochemistry. Am J Surg Pathol. 2001;25(9):

1208–1210.

410. Wieczorek TJ, Pinkus JL, Glickman JN, et al. Comparison of

thyroid transcription factor-1 and hepatocyte antigen immuno-

histochemical analysis in the differential diagnosis of hepatocel-

lular carcinoma, metastatic adenocarcinoma, renal cell

carcinoma, and adrenal cortical carcinoma. Am J Clin Pathol.

2002;118(6):911–921.

411. Wiedorn KH, Goldmann T, Henne C, et al. EnVisionþ, a new

dextran polymer-based signal enhancement technique for in situ

hybridization (ISH). J Histochem Cytochem. 2001;49(9):

1067–1071.

412. Williams JH, Mepham BL, Wright DH. Tissue preparation for

immunocytochemistry. J Clin Pathol. 1997;50(5):422–428.

413. Willingham MC. Conditional epitopes: is your antibody always

specific? J Histochem Cytochem. 1999;47(10):1233–1235.

414. Wilson E, Jackson S, Cruwys S, et al. An evaluation of the

immunohistochemistry benefits of boric acid antigen retrieval

on rat decalcified joint tissues. J Immunol Methods. 2007;

322(1–2):137–142.

415. Wilson JE. The use of monoclonal antibodies and limited

proteolysis in elucidation of structure-function relationships in

proteins. Methods Biochem Anal. 1991;35:207–250.

416. Wolff AC, Hammond MEH, Scwartz JN, et al. American Soci-

ety of Clinical Oncology/College of American Pathologists

guideline recommendations for human epidermal growth factor

receptor 2 testing in breast cancer. J Clin Oncol. 2007;25(1):

118–145.

417. Wong SCC, Chan JKC, Lo ESF, et al. The contribution of

bifunctional SkipDewax pretreatment solution, rabbit monoclo-

nal antibodies, and polymer detection systems in immunohisto-

chemistry. Arch Pathol Lab Med. 2007;131(7):1047–1055.

418. Wood GS, Warnke R. Suppression of endogenous avidin-

binding activity in tissues and its relevance to biotin-avidin

detection systems. J Histochem Cytochem. 1981;29(10):

1196–1204.

419. World Organization for Animal Health (OIE). Principles and

methods of validation of diagnostic assays for infectious

diseases. In: OIE Manual of Diagnostic Tests and Vaccines for

Terrestrial Animals. 7th ed. Paris, France: OIE; 2012:34–51.

420. Xie R, Chung JY, Ylaya K, et al. Factors influencing the

degradation of archival formalin-fixed paraffin-embedded tissue

sections. J Histochem Cytochem. 2011;59(4):356–365.

421. Yamashita S. Heat-induced antigen retrieval: mechanisms and

application to histochemistry. Prog Histochem Cytochem.

2007;41(3):141–200.

422. Yamashita S, Okada Y. Mechanisms of heat-induced antigen

retrieval: analyses in vitro employing SDS-PAGE and immuno-

histochemistry. J Histochem Cytochem. 2005;53(1):13–21.

423. Yamashita S, Okada Y. Application of heat-induced antigen

retrieval to aldehyde-fixed fresh frozen sections. J Histochem

Cytochem. 2005;53(11):1421–1432.

424. Yamazaki F, Aragane Y, Kawada A, et al. Immunohistochem-

ical detection for nuclear b-catenin in sporadic basal cell carci-

noma. Br J Dermatol. 2001;145(5):771–777.

425. Yaziji H, Barry T. Diagnostic immunohistochemistry: what can

go wrong? Adv Anat Pathol. 2006;13(5):238–246.

426. Yaziji H, Taylor CR, Goldstein NS, et al. Consensus recommen-

dations on estrogen receptor testing in breast cancer by immuno-

histochemistry. Appl Immunohistochem Mol Morphol. 2008;

16(6):513–520.

427. Yelton DE, Scharff MD. Monoclonal antibodies: a powerful new

tool in biology and medicine. Ann Rev Biochem. 1981;50:

657–680.

428. Yokoyama S, Kashima K, Inoue S, et al. Biotin-containing intra-

nuclear inclusions in endometrial glands during gestation and

puerperium. Am J Clin Pathol. 1993;99(1):13–17.

429. Yoshie S, Hagn C, Ehrhart M, et al. Immunological characteri-

zation of chromogranins A and B and secretogranin II in the

bovine pancreatic islet. Histochemistry. 1987;87(2):99–106.

430. Young AN, Kairdolf BA. Nanotechnology in molecular diagnos-

tics. In: Cheng L, Zhang DY, Eble JN, eds. Molecular Genetic

Pathology. New York, NY: Springer; 2013:383–398.

431. Yu B, Liu YJ, Yin X. Elimination of non-specific staining in

immunohistochemistry of human liver tissues by modulation

of antibody-antigen binding microenvironment. J Histotechnol.

2013;36(1):3–10.

432. Zbaeren J, Solenthaler M, Schaper M, et al. A new fixative allow-

ing accurate immunostaining of kappa and lambda immunoglobu-

lin light chain expressing B-cells without antigen retrieval in

paraffin-embedded tissue. J Histotechnol. 2004;27(2):87–92.




433. Zeheb R. Automating immunohistochemistry. In: Key M, ed.

Immunohistochemical Staining Methods. 4th ed. Carpinteria,

CA: Dako Corporation; 2006:103–106.

434. Zhang PJ, Wang H, Wrona EL, et al. Effects of tissue fixatives

on antigen preservation for immunohistochemistry: a compara-

tive study of microwave antigen retrieval on Lillie fixative and

neutral buffered formalin. J Histotechnol. 1998;21(2):101–106.

435. Zhang Z, Zhao L, Guo H, et al. Diagnostic significance of immu-

nocytochemistry on fine needle aspiration biopsies processed by

thin-layer cytology. Diagn Cytopathol. 2012;40(12):1071–1076.

436. Zhong X, Lizardi PM, Huang X, et al. Visualization of oligonu-

cleotide probes and point mutations in interphase nuclei and

DNA fibers using rolling circle DNA amplification. Proc Natl

Acad Sci U S A. 2001;98(7):3940–3945.




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