Importance Of Secondary Antibody Choice In ... · Importance Of Secondary Antibody Choice In Immunohistochemistry: Learning From A Failure Corresponding Author: Prof. Jeanne A Pawitan,

Article ID: WMC001453 2046-1690

Importance Of Secondary Antibody Choice InImmunohistochemistry: Learning From A FailureCorresponding Author:Prof. Jeanne A Pawitan,Author, Dept. of Histology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia, Jl. Tanjung 48,10430 - Indonesia

Submitting Author:Prof. Jeanne Adiwinata Pawitan,Professor, Dept. of Histology, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia, 10430 - Indonesia

Article ID: WMC001453

Article Type: Research articles

Submitted on:06-Jan-2011, 02:37:37 AM GMT Published on: 07-Jan-2011, 12:11:05 PM GMT

Article URL: http://www.webmedcentral.com/article_view/1453

Subject Categories:IMMUNOHISTOCHEMISTRY

Keywords:immunohistochemistry, paraffin sections, primary antibody, secondary antibody, Tenascin C, falsepositive, false negative

How to cite the article:Antarianto R D, Pawitan J A, Jusuf A A. Importance Of Secondary Antibody Choice InImmunohistochemistry: Learning From A Failure . WebmedCentral IMMUNOHISTOCHEMISTRY2011;2(1):WMC001453

Source(s) of Funding:

The funding of this study was supported by the university of Indonesia Master’s degree Research Grant, contractnumber: 243C/DRPM-UI/N1.4/2008

Competing Interests:

Competing interest none declared

WebmedCentral > Research articles Page 1 of 6

http://www.webmedcentral.com/article_view/1453

WMC001453 Downloaded from http://www.webmedcentral.com on 24-Dec-2011, 11:24:37 AM

Importance Of Secondary Antibody Choice InImmunohistochemistry: Learning From A FailureAuthor(s): Antarianto R D, Pawitan J A, Jusuf A A

Abstract

Aim: to optimize Tenascin-C immunohistochemistrystaining protocol for rat cardiac tissueMethods: This is a preliminary experimental study atHistology Lab Faculty of Medicine University ofIndonesia from 7th December 2007 to 2nd July 2008.Samples were paraffin sections of rat cardiac tissues,and paraffin sections of brain tissue were used aspositive control and absence of overnight primaryantibody incubation as negative control. Formalinfixation was used before paraffin blocks were made.Immunohisto-chemistry protocol was modified fromHannekamp, et al, who stained human tissue.Stainingresults were recorded and analyzed. Staining wasconsidered successful when the positive and negativecontrols were positive and negative respectively. If theprotocol was considered unsuccessful, the dataobtained and the steps in the protocol was reviewedand analyzed for cause/s o f fa i lure andtroubleshooting steps were taken.Results: The staining results according to the protocoland subsequent troubleshooting protocols showedmostly false positive results, which cause wassupposed to be the cross reaction of secondaryantibody with the rat tissue.Conclusion: In starting a new immunohistochemistrystaining for paraffin sections, care should be paid onthe compatibility of the kit for paraffin section, choiceof primary antibody that react with the antigen to bestudied, and choice of secondary antibody that do notreact with the tissue to be studied.

Introduction

Immunohistochemistry technique is the visualization ofa tissue or cellular component in situ by detectingspecific antigen using antibody-antigen interactionswhere the antibody has been tagged with a visiblemarker. The marker may be a fluorescent dye,colloidal metal, hapten, radioactive marker or anenzyme that digest a substrate to reveal the substratecolor.Optimal visualization of a certain protein occurs when

higher signal to non-specific background (noise) ratiois achieved. Therefore, signal amplification andreduction of non-specific background staining is theultimate strategy to obtain optimal result.Signal amplification may be performed using labeledStreptAvidin-Biotin (LSAB). In this method, there arethree reactions. The first reaction is between anon-labeled primary antibody and specific antigen intissues/cells. The second is between the primaryantibody and a biotinylated secondary antibody, andthe final reaction is between biotin and horse radishp e r o x i d a s e - c o n j u g a t e d S t r e p t a v i d i n(HRP-StreptAvidin). The peroxidase will then oxidize achromogen substrate that will appear as a brownishsubstance.Tenascin-C (TNC) is an extracellular matrixglycoprotein that may be found in stem cellmicroenvironment (stem cell niche) at bone marrowand subventricular zone of the brain and possibly atother organ as well [1, 2]. Tenascin-C was firstisolated from embrionic tissue, while in adult tissueTNC expression remains at epithelial-mesenchymaljunction. Tenascin C is known as regulator for severalpost-natal cellular functions, e.g: cell growth, T cellsuppression, and wound healing [3]. Other than that,TNC also play a role in hemmaglutination,inflammation and progression towards invasive cancer.These findings harbored the significance of tenascin Cexpression at tissue hotspots, making it an interestingprotein to investigate. In order to begin studies ontenascin C expression, we first aimed to optimizeTenascin-C immunohistochemistry staining protocolfor rat cardiac tissue.MethodsThis is a preliminary experimental study to optimizeTNC immunohistochemistry staining, performed atHistology Lab Faculty of Medicine University ofIndonesia from 7th December 2007 to 2nd July 2008.Samples were paraffin sections of rat cardiac tissues,and paraffin sections of brain tissue were used aspositive control and absence of overnight primaryantibody incubation as negative control. Formalinfixation was used before paraffin blocks were made.Protocol of the whole study was approved by EthicalCommission for animal studies Ministry of HealthRepublic of Indonesia number LB.03.02/KE/1529/2008.Immunohistochemistry protocol was modified fromHannekamp, et al [4].Staining results were recordedand analyzed. Staining was considered successful



when the positive and negative controls were positiveand negative respectively. If the protocol wasconsidered unsuccessful, the data obtained and thesteps in the protocol was reviewed and analyzed forcause/s of failure and troubleshooting steps weretaken.Immunohistochemistry staining protocolProtocol for immunohistochemistry staining procedureincluded de-paraffinization, rehydration, blocking ofendogenous peroxidase activity using hydrogenperoxide, antigen retrieval, blocking of non-specificbackground, treatment with primary antibody,secondary antibody, visualization of antigen-antibodyreaction, counterstaining and mounting.De-paraffinization and re-hydrationThe parafin sections were placed on to glass slide,and deparaffinisation was done by immersing theglass slide in xylene 2 times for 5 minutes each.Subsequent rehydration in graded alcoholconcentration was done using absolute alcohol,alcohol 95% and alcohol 80%; each for 5 minutes.Blocking of endogenous peroxidase activityBlocking of endogenous peroxidase activity was doneinside a moist chamber. Every slide was covered with4% v/v H2O2 in methanol for 20 minutes, followed bywashing in running tap water for 3 minutesAntigen retrievalThe slides were placed inside a Coplin jar that wasfilled with citrate buffer pH 6.0 and heated in amicrowave until reaching boiling point for 5 minutes,and the heating was repeated three times. Then theslides were let to cool in room temperature for 45minutes, and wash by repeated dipping in PBS pH 7.4three times for 3 minutes each.Blocking of non-specific backgroundBlocking serum: blocking of non-specific backgroundstaining was done using normal rabbit serum (10% v/vserum in PBS pH 7.4 for 20 minutesTreatment with primary antibody and secondaryantibodyDilution of primary antibody: anti-human TNC (N19)goat IgG (Santa Cruz sc9871), 1:200 was done in PBSpH 7.4 mixed with 1% v/v blocking serum. Fortreatments with antibodies, the slides were placed in amoist chamber. Primary antibody treatment was doneby incubating the slides with diluted primary antibodyat 40 C overnight. The next day, the slides werewashed in PBS pH 7.4 twice for 3 minutes each.Incubation with secondary antibody (biotinylatedanti-goat swine IgG, LSAB kit, Dako k-0679) was doneat room temperature for 15-30 minutes. After that,excess solution was wiped off from the slides. Visualization of antigen-antibody reactionVisualization of antigen-antibody reaction was done by

incubation with streptavidin-HRP for 15-30 minutes,then the slides were washed with PBS pH 7.4 twice for3 minutes each, and excess solution was wiped offfrom slides. Further, incubation with DAB chromogensubstrate was done at room temperature for 10minutes, followed by washing with PBS pH 7.4, andthen with running tap water.Counterstaining and mountingCounterstaining was done with Lily Mayer’sHematoxylin for 2 minutes, followed by step wisedehydration using graded alcohol concentration: 95%and absolute alcohol twice, each for 5 minutes. Afterthat, clearing was done twice with Xylene for 5minuteseach, and finally the slides were mounted with Entelan.Analysis of dataWhen false positive results were obtained: theprocedure was reviewed for possible causes, i.e.antibodies were too concentrated and washing wasnot optimal, peroxide treatment was not optimal eitherin the concentration of the peroxide, time of incubation,or the peroxide had lost its activity, or blocking of nonspecific background was not optimal.When false negative results were obtained: theprocedure was reviewed for possible causes, i.e.de-paraffinization and re-hydration was not optimal,antigen retrieval was not optimal, dilution of primaryantibody was over diluted, or incubation time ofprimary antibody was too short.After the protocol was reviewed, and cause of failuresupposed, subsequent modifications of protocol weremade. Staining results from the protocol andsubsequent modifications were recorded andcompared to obtain the optimized protocol.ResultsThe staining results according to the protocol showedpositive staining on all of the slides, including thenegative control (false positive). Troubleshooting todevelop subsequent procedure was done, and overall,there were 4 modifications of the protocol (Table 1).Result of modification 1 was false negative. However,staining of cardiac tissue showed inconsistent staining,and some showed positive staining on cytoplasm andnucleus of cardiomyocytes (intracellular components).Further, modification 2-4 mostly showed inconsistentresults with mainly false positive results. As mostly ofthe staining gave false positive results, we checkedthe specification of secondary antibody, and we foundout that the secondary antibody in LSAB kitDako-0679 (anti-goat swine IgG) is a non- specificantibody as it can cross react with rabbit and mouseIgG.DiscussionIn this study, the primary antibody was goat IgGagainst human TNC, which cross react to mouse and



rat TNC according to the manufacturer. Though theprimary ant ibody is not recommended forimmunohistochemistry staining, it was used byHanekamp et al for immunohistochemistry staining ofhuman tissue with good result [5].When the protocol according to modified protocol ofHannekamp et al yielded false positive result, wesupposed that the in-optimal blocking of endogenousperoxidase activity and nonspecific background wasthe cause. Therefore, in modification-1 protocol, wechange the peroxidase with that from the kit, and theblocking serum with normal horse serum. In addition,we use de-ionized instead of tap water for the washingstep, and we got false negative results. However,some of the cardiac tissue showed positiveintracellular staining. At the time of experiment, wesupposed that the positive staining was nonspecificbackground (noise), as a previous study showedtenascin-C as extracellular/ intercellular molecules [5],though in our later finding using Santa Cruz staining kitthat gave good result and consistent Tenascin Cstaining in rat tissue, we found tenascin C expressionin cardiomyocytes [6]. We supposed the false negativeresult was due to improper de-paraffinization andre-hydration that caused in-optimal antigen retrievaland failure in antigen-antibody reaction. In addition,nonspecific background may be due to highconcentration of primary antibody. Therefore, inmodif icat ion-2 protocol we elaborate thede-paraffinization and dehydration steps, but wereduced the amount of primary antibody, and indeedwe got positive results, but also on the negativecontrols.We supposed that the false positive resultwas due to prolonged exposure to the primaryantibody, which caused insufficient washing, and traceprimary antibody was still found on the specimen.Therefore, in modification-3 and -4protocol we usedthe first concentration of primary antibody and weeither reduced the time to 30 minutes or overnight, butand we still got mostly false positive results. As themost of our result showed false positive result, wesupposed that the cause was the secondary antibodythat we did not suspect before. Therefore, in starting anew immunohistochemistry staining, not only theprimary antibody and the compatibility of the kit forparaffin section should be taken into account, but alsothe nature of secondary antibody. In this preliminarystudy, we have been not careful in choosing the kit,and not careful in reading the property of the kit. Ourcareless made us used a kit that contained asecondary antibody that according to the manufacturercross reacts with mouse tissue, while the animal weused are rat; so the secondary antibody may reactwith rat tissue as well, thus the mostly false positive

results.conclusionIn starting a new immunohistochemistry staining forparaffin sections, care should be paid on thecompatibility of the kit for paraffin section, choice ofprimary antibody that react with the antigen to bestudied, and choice of secondary antibody that do notreact with the tissue to be studied.

References

1. Yanagisawa M, Yu RK. The expression andfunctions of glycoconjugates in neural stem cells.Glycobiology. 2007;17(7):57R-74R.2. Jones FS, Jones PL. The Tenascin family of ECMglycoproteins: Structure, function, and regulationduring embryonic development and tissue remodeling.Developmental Dynamics 2000; 218:235–593. Ballard VL, Sharma A, Duignan I, Holm JM, Chin A,Choi R, Hajjar KA, Wong SC, Edelberg JM. Vasculartenascin-C regulates cardiac endothelial phenotypeand neovascularization. FASEB J. 2006; 20: 717–9.4. Hanekamp EE, Gielen SC, Smid-Koopman E,Kühne LC, de Ruiter PE, Chadha-Ajwani S,Brinkmann AO, Grootegoed JA, Burger CW,Huikeshoven FJ, Blok LJ. Consequences of loss ofprogesterone receptor, expression in development ofinvasive endometrial cancer. Clin Cancer Res. 2003;9:4190-9.5. Tamaoki M, Imanaka-Yoshida K, Yokoyama K,Nishioka T, Inada H, Hiroe M, Sakakura T, Yoshida T.Tenascin-C Regulates Recruitment of Myofibroblastsduring Tissue Repair after Myocardial Injury. Am JPathol. 2005; 167(1): 71–80.6. Antarianto R, Pawitan JA, Jusuf AA. Expression oftenascin C in rat neonatal cardiac tissue. RegenerativeMedizin 2008;1:95. Abstract.



Table 1. Procedure according to the protocol and the subsequent procedures

Treatment Concentration Time Repeat Trouble shooting

Xylene 1x 5 minutes 2x M2-4: 10 minutes

Alcohol 95%, 80% 5 minutes 1x M2-4: 95%, 90%,80%,70%,50%

Peroxide (H2O2) 4% v/v in methanol 20 minutes 1x M1-4: Endogenperoxidase blockingsolution (kit LSABDako)

Washing: running tapwater

1x 3 minutes 1x M1-4: de-ionizedwater

Citrate buffer pH 6.0

At boiling point

1x 5 minutes 3x

Blocking serum:normal rabbit serum

10% v/v in PBS 20 minutes 1x M1-4: normal horseserum

Primary Ab: anti-TNCgoat IgG (santa Cruzsc9871), at 40 C

1:200 in PBS-blocking serum 1%

Overnight 1x M2: 1:400 in PBS-blocking serum 1%

M3: 30 minutes

Washing: PBS pH 7.4 1x 3 minutes 2x

Secondary Ab:biotinylated anti-goatswine IgG (LSAB kit,Dako k-0679), at roomtemperature

1x 15-30minutes

1x

Alcohol 95% 5 minutes 1x M2-4: 50%, 70%,80%, 90%, 95%

Alcohol 100% 5 minutes 2x

Xylene 100% 5 minutes 2x M2: 10 minutes

Ab= antibody, IgG= immunoglobulin G, TNC= tenascin C, LSAB= LabeledStreptAvidin-Biotin, v/v= volume per volume, M1= modification 1, M2= modification 2,M3= modification 3, M4= modification 4

Illustrations

Illustration 1

Table 1



DisclaimerThis article has been downloaded from WebmedCentral. With our unique author driven post publication peerreview, contents posted on this web portal do not undergo any prepublication peer or editorial review. It iscompletely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscriptbut also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions beforesubmitting any information that requires obtaining a consent or approval from a third party. Authors should alsoensure not to submit any information which they do not have the copyright of or of which they have transferredthe copyrights to a third party.

Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater tothe needs of an individual patient. The web portal or any content(s) therein is neither designed to support, norreplace, the relationship that exists between a patient/site visitor and his/her physician. Your use of theWebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harmthat you may suffer or inflict on a third person by following the contents of this website.


Documents

Importance Of Secondary Antibody Choice In ... · Importance Of Secondary Antibody Choice In Immunohistochemistry: Learning From A Failure Corresponding Author: Prof. Jeanne A Pawitan,