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3/17/2012
1
STAINING
STAINING
• The process of applying dyes on the sections to study
architectural pattern of the tissue and physical
characteristics of the cells.
• Different tissues and cells have varying affinities for
most dyes and stains
AFFINITY:
Acidic (nucleus) >>>>>>> basic stains
Basic (cytoplasm) >>>>>>> acidic stains
MAJOR GROUPS OF TISSUE STAINING
1. HISTOLOGICAL STAINING
The process whereby the tissue constituents are
demonstrated in sections by direct interaction with a
dye or staining solution
Active tissue component is colored
E.g. micro-anatomical stains, bacterial stains, specific
tissue stains (e.g. muscles, connective tissue and
neurologic stains)
2. HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
The process whereby various constituents of tissues are studied thru chemical reactions that permits microscopic localization of specific tissue substances
E.g. Perl’s prussian blue reaction for hemoglobin and Periodic Acid Schiff staining for carbohydrates
MAJOR GROUPS OF TISSUE STAINING
Enzyme histochemistry Active reagent - substrate
Tissue - enzymes
The final opacity or coloration is produced from the
substrate rather than the tissue.
3. IMMUNOHISTOCHEMICAL STAINING A combination of immunologic and histochemical
techniques that allow phenotypic markers to be detected
by antibodies (e. g. polyclonal, monoclonal, enzyme-
labeled or fluorescent-labeled)
MAJOR GROUPS OF TISSUE STAINING
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METHODS OF STAINING
1. Direct staining
Uses aqueous or alcoholic dye solutions (e.g.
methylene blue, eosin) to produce a color
2. Indirect staining
Uses a mordant or another agent to intensify
the action of the dye used
2. Indirect staining
MORDANT
Serves as a link or bridge between the tissue and the
dye
The dye may stain weakly by itself, therefore the
mordant combines with the dye forming a colored “lake”
which would combine with the tissue forming an
insoluble “tissue-mordant-dye-complex”, which would
allow subsequent counterstaining and dehydration
E.g. Potassium alum with hematoxylin in Ehrlich’s
hematoxylin. Iron in Weigert’s hematoxylin
METHODS OF STAINING
2. Indirect staining
ACCENTUATOR
Not essential and does not participate to the chemical
reaction of the tissue and dye
Accelerates the speed of the staining reaction by
increasing the staining power and selectivity of the dye
E.g. Potassium hydroxide in Loeffler’s methylene blue,
Phenol in Carbol thionine and Carbol fuchsin
METHODS OF STAINING PROGRESSIVE STAINING
Tissue elements are stained in definite
sequence
The staining with specific periods of time or
until desired color is attained
Not washed or decolorized
The distinction of tissue detail relies solely on
the selective affinity of the dye for various
cellular elements
REGRESSIVE STAINING
First over-stain the tissue to obliterate cellular
details
Excess stain is removed or decolorized from
unwanted parts of the tissue and until the
desired color is obtained
DIFFERENTIATION / DECOLORIZATION
The selective removal of excess stain from the tissue during regressive staining so that a specific subatance may stain distinctly from the surrounding tissue
Usually done by washing the section in simple solution (e.g. water or alcohol) or use of acids and oxidizing agents
Primary stain = basic dye
Differentation = acidic solution
Primary stain = acidic dye
Differentation = alkaline solution
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DIFFERENTIATION / DECOLORIZATION
Alcohol
Differentiator for both acidic and basic dyes by dissolving excess dye
Mordant (e.g. iron alum)
A differentiating agent
Can oxidize hematoxylin to a soluble, colorless compound.
Disadvantage: if a mordant stained section is allowed to remain in a differentiating agent such as 1% or 2% alcohol, all of the dye will be removed. Restaining faded slides
METACHROMATIC STAINING
Makes use of specific dyes which differentiate
particular substances by staining it with a color that is
different from that of the stain itself (metachromasia)
Usually employed in staining cartilage, connective
tissue, epithelial mucins, amyloid and mast cell
granules.
Metachromatic dyes (basic) – belongs to thizine and triphenylmethane groups
1. Methyl violet or crystal violet
2. Cresyl blue (for reticulocytes)
3. Safranin
4. Bismarck brown
5. Basic fuchsin
6. Methylene blue
7. Thionine
8. Toluidine blue
9. Azure A, B, C
METACHROMATIC STAINING
Water is necessary for most metachromatic staining techniques
Metachromasia is usually lost if section is dehydrated in alcohol after staining.
Metachromasia is satisfactorily seen in formalin-fixed tissues.
METACHROMATIC STAINING
COUNTERSTAINING
application of a different color or stain to provide contrast
and background to the staining of the structural
components to be demonstrated.
Cytoplasmic stains
Red Yellow Green
Eosin Y Picric acid Lt. Green SF
Eosin B Orange G Lissamine Green
Phloxine B Rose Bengal
COUNTERSTAINING
Nuclear stains
Red Blue
Neutral red Methylene blue
Safranin O Toluidine blue
Carmine Celestine blue
Hematoxylin
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METALLIC IMPREGNATION
The process where specific tissue elements are
demonstrated not by stains but by colorless solutions of
metallic salts which are deposited on the surface of the
tissue
It is not absorbed by the tissues, could be a precipitate
or a reduction product on certain tissues
E.g. Gold chloride, Silver nitrate
VITAL STAINING
The selective staining of living cell constituents
Demonstrates cytoplasmic structures
By engulfment of the dye particle
By staining of pre-existing cellular components
Nucleus is resistant to vital stains
VITAL STAINING
Two types
1. Intravital staining
by injecting the dye into any part of the animal body
e.g. lithium, carmine and India ink
2. Supravital staining
used immediately after removal of cells from the living
body
e.g. Neutral red (best), Janus green (mitochondria), Trypan
blue, Nile blue, Thionine and Toluidine Blue
Most common method utilized for microanatomical
studies of tissues
Uses the regressive staining which consists of
a. overstating the nuclei
b. removal of superfluous and excessive
color of the tissue constituent by acid differentiation
Routine Hematoxylin and Eosin (H & E)
H and E PROCEDURE
1. Clear paraffin embedded sections in first xylene bath for 3 minutes
2. Transfer to second xylene bath for 2 to 3 minutes.
3. Immerse in first bath of absolute ethyl alcohol for 2 minutes.
4. Transfer to a bath of 95% ethyl alcohol for 1 to 2 minutes.
5. Rinse in running water for a minute.
6. Stain with Harris Alum Hematoxylin for 5 minutes (Ehrlich’s hematoxylin requires 15-30 minutes).
7. Wash in running tap water to remove excess stain.
8. Differentiate in 1% acid-alcohol (1mL conc. HCl to 99 mL of 80% ethyl alcohol) for 10-30 secs. Until the nuclei are stained.
9. Rinse in tap water.
10. Use ammonia water (average of 5 minutes) or 1% aqueous lithium carbonate until the section appears blue (about 30 seconds)
H and E PROCEDURE
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11. Wash in running water for 5 minutes.
12. Counterstain with 5% aqueous eosin for 5 minutes. If alcoholic eosin is used, the time can be reduced to 30 seconds or 1 minute.
13. If aqueous eosin is used, wash and differentiate in tap water under microscopic control until the nuclei appear sharp blue to blue black and the rest is in shades of pink. If alcoholic solution is used, differentiate with 70% alcohol.
14. Dehydrate, clear and mount.
H and E PROCEDURE
Nuclei – blue to blue black
Karyosome – dark blue
Cytoplasm – pale pink
RBCs, eosinophilic granules, keratin – bright-
orange red
Calcium and decalcified bone –purplish blue
Decalcified bone matrix, collage and osteoid –
pink
Muscle fibers – deep pink
H and E RESULT
STAINS AND STAINING
SOLUTIONS
STAINS AND STAINING SOLUTIONS
Divided into 2 categories:
1.Natural dyes
2.Synthetic (Artificial) dyes
STAINS AND STAINING SOLUTIONS
NATURAL DYES
Derived from plants and animals
E.g. Hematoxylin, Cochineal dyes, Orcein,
Saffron
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STAINS AND STAINING SOLUTIONS
NATURAL DYES
1. Hematoxylin
Hematoxylin campechianum
It is not a stain
Active coloring agent – hematin
Oxidation of hematoxylin through the process of
“ripening”
Used in combination with a mordant such as
alum, iron, chromium and copper salts
STAINS AND STAINING SOLUTIONS
NATURAL DYES
1. Hematoxylin
Ripening
Natural ripening
Expose the substance to air and sunlight
A slow process, 3-4 months
Artificial ripening
Chemical oxidation
Hydrogen peroxide, mercuric oxide, potassium permanganate,
Sodium perborate, sodium iodate
Over-ripening
Excessive oxidation
STAINS AND STAINING SOLUTIONS
NATURAL DYES
2. Cochineal dyes
Extracted from Coccus cacti
With alum
Carmine dye
Chromatin and nuclear stain for fresh and smear preparation
With picric acid
Picrocarmine
Neuropathological stain
With aluminum chloride
Best’s carmine
Demonstration of glycogen
STAINS AND STAINING SOLUTIONS
NATURAL DYES
3. Orcein
Vegetable dye
Extracted from lichens
Colorless, treated with ammonia, exposed to air to
produce a blue or violet color
Weak acid, soluble in alkali
Used for staining elastic fibers
STAINS AND STAINING SOLUTIONS
SYNTHETIC DYES Also known as “coal tar dyes”
Derived from hydrocarbon benzene
Collectively known as “aniline dye”
Chromophore
Substances that are capable of producing visible color but is not
permanent and can be easily removed
Auxochrome Substances that are added to a chromogen, which alters the
property of the chromogen by altering its shade, enabling it to form
salts with another compound and enables it to retain its color in the
tissue
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STAINS AND STAINING SOLUTIONS
SYNTHETIC DYES Classified into 3 groups based on where the coloring
substance is found
1. Acid dyes
The coloring substance is found in the acid component
and the inactive base is usually the sodium salt of a
sulfonate of rosaniline
Basic cell structures have an affinity for acid dye ions
and are called acidophilic
STAINS AND STAINING SOLUTIONS
SYNTHETIC DYES Classified into 3 groups based on where the coloring
substance is found
2. Basic dyes
The coloring substance is found in the basic component
that combines with the acid radical
Acidic structures have an affinity for basic dyes and are
called basophilic
STAINS AND STAINING SOLUTIONS
SYNTHETIC DYES Classified into 3 groups based on where the coloring
substance is found
3. Neutral dyes
Formed by combining aqueous solutions of acid and
basic dyes
Stains the cytoplasm and nucleus simultaneously and
differentially
Insoluble to barely soluble in water
Soluble in alcohol
STAINS AND STAINING SOLUTIONS
COMMON STAINING SOLUTIONS 1. Hematoxylin
Most commonly used for histologic studies
A. Aluminum hematoxylin
Progressive and regressive staining
2 main alum hematoxylin (ripening agent) Erlich’s – sodium iodate
Harris – mercuric chloride
Forms blue lakes
STAINS AND STAINING SOLUTIONS
COMMON STAINING SOLUTIONS 1. Hematoxylin
B. Erlich’s Hematoxylin
Natural ripening hematoxylin ( 2 months)
Regressive staining
Sodium Iodate shortens lifespan
Stains mucopolysaccharide substances (e.g. cartilages,
cement lines of bones), tissues subjected in acid
decalcification, tissues stored in formalin
Not an ideal stain for frozen sections
STAINS AND STAINING SOLUTIONS
COMMON STAINING SOLUTIONS 1. Hematoxylin
C. Harris Hematoxylin
Dark purple color when ripened with mercuric chloride
Glacial acid for better nuclear staining
Routine stain used in
D. Cole’s Hematoxylin Ripened with alcoholic iodine
E. Mayer’s Hematoxylin
Ripened with sodium iodate
Nuclear counterstain
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STAINS AND STAINING SOLUTIONS
COMMON STAINING SOLUTIONS 2.
Iron Hematoxylin
Used for regressive staining
Blue black lakes
Iron is an active oxidizing agent
STAINS AND STAINING SOLUTIONS
COMMON STAINING SOLUTIONS 2. Iron Hematoxylin
A. Weigert’s Hematoxylin
Standard iron hematoxylin
Used in demonstrating connective tissue
B. Heidenhain’s Hematoxylin For nuclear and cytoplasmic inclusions
C. Phosphotongstic Acid Hematoxylin
Natural ripening achieved with light and air
Color: reddish brown to purple
Progressive stain
STAINS AND STAINING SOLUTIONS
COMMON STAINING SOLUTIONS 3. Eosin
A red acid dye
Routinely used as a counterstain after hematoxylin and
before methylene blue
Stains connective tissues and cytoplasm differentially
3 forms:
Yellow (Eosin Y)
Most commonly used
Green yellow fluorescence
STAINS AND STAINING SOLUTIONS
COMMON STAINING SOLUTIONS 3. Eosin
3 forms:
Eosin B, Erythrosin B
Deeper red color
Eosin S, Eosin alcohol-soluble
Ethyl eosin
STAINS AND STAINING SOLUTIONS
OTHER STAINS1. Acid Fuchsin-Picric Acid (Van Gieson’s Stain)
For demonstration of connective tissues
2. Acridine orange (Masson Stain)
Basic acridine fluorochrome
Discriminates dead and living cells
DNA – green fluorescence
RNA – red fluorescence
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STAINS AND STAINING SOLUTIONS
OTHER STAINS3. Acridine Red 3B
Demonstration of calcium salt deposits and Phosphatase
activities
4. Alcian Blue
Water soluble phthalocyanin dye
For connective tissue and epithelial mucin
5. Aniline Blue
Cytoplasmic stain
For counterstaining of epithelial sections
STAINS AND STAINING SOLUTIONS
OTHER STAINS6. Basic Fuchsin
Plasma stain
For staining acid-fast organisms, for mitochondria, for
differentiation of smooth muscles (with the use of picric acid)
Feulgen’s and Schiff’s reagent for detection of aldehydes
Van Gieson’s solution for staining connective tissues, mucin
and elastic tissues
STAINS AND STAINING SOLUTIONS
OTHER STAINS6. Basic Fuchsin
A. Carbol-Fuchsin
B. Coleman’s Feulgen Reagent
C. Schiff’s Reagent
D. Mallory’s Fuchsin Stain
E. Aldehyde Fuchsin (Gomori’s stain)
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STAINS AND STAINING SOLUTIONS
OTHER STAINS7. Benzidine
For staining hemoglobin
8. Bismarck Brown
Used as counterstain for Gram’s technique, for acid fast, for
Papanicolau method
Used for staining diphtheria organisms
STAINS AND STAINING SOLUTIONS
OTHER STAINS9. Carmine
Used as chromatin stain for fresh materials in smear
preparations
Slightly soluble in water and kept in ammoniacal solution
Combined with aluminum chloride to stain glycogen (Best
Carmine solution)
10. Celestine Blue
Used for routine staining of fixed sections
Resistant to strong acid dyes
Good nuclear stain
STAINS AND STAINING SOLUTIONS
OTHER STAINS11. Congo Red
Best known as an indicator
Stains elastic tissues, amyloid, myelin
12. Crystal Violet
A nuclear or chromatin stain
Stains amyloid in frozen sections, platelets in blood
Gentian violet (crystal violet, methyl violet, dexterin)
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STAINS AND STAINING SOLUTIONS
OTHER STAINS13. Giemsa Stain
Used for staining blood to differentiate WBCs
14. Gold Sublimate
Stain used for metallic impregnation
Made up of gold chloride and mercuric chloride
STAINS AND STAINING SOLUTIONS
OTHER STAINS15. Iodine
Stains amyloid, cellulose, starch, carotenes, glycogen
Used to remove mercuric fixative artifact pigments
Used as a reagent to alter crystal and methyl violet which may
be retained by certain bacteria and fungi
Gram’s iodine Stains microorganisms and fibrin in tissue sections
Lugol’s iodine Used as test for glycogen, amyloid and corpora amylacea
STAINS AND STAINING SOLUTIONS
OTHER STAINS16. Janus Green B
For demonstrating mitochondria during intravital staining
17. Malachite Green
Counterstain for ascaris eggs, erythrocytes, bacterial spore
stain
Used as a decolorizer and counterstain
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STAINS AND STAINING SOLUTIONS
OTHER STAINS18. Methylene Blue
Common basic nuclear stain used with eosin
Stains plasma cells, cytological examination of sputum for
malignant cells, evaluation and differentiation of bacteria,
diagnosis of diphtheria, vital staining of nervous tissues
19. Neutral Red
Basic stain
For demonstration of cell granules and vacuoles of phagocytic
cells
STAINS AND STAINING SOLUTIONS
OTHER STAINS20. Orcein
Stains elastic fibers
Recommended for dermatological studies Demonstrates the finest and most delicate fibers in the skin
21. Osmium Tetroxide
Used to stain fat – black
STAINS AND STAINING SOLUTIONS
OTHER STAINS22. Picric Acid
Counterstain for acid fuchsin, connective tissues (in Van
Gieson’s stain), cytoplasmic stain in contrast to basic dyes,
counterstain for crystal violet
23. Prussian Blue
Colored salt of ferric ferrocyanide
Used for the manufacture of paints
Used as contrast stain, intravital staining of the circulatory
system
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STAINS AND STAINING SOLUTIONS
OTHER STAINS24. Rhodamine B
Used with osmic acid to fix and stain blood and glandular
tissues
25. Silver Nitrate
Used for identification of spirochetes, reticulum, fiber stains
26. Toluidine Blue
Used as nuclear stain in fixed tissues, stains Nissl granules or
chromophilic bodies
STAINS AND STAINING SOLUTIONS
OIL SOLUBLE DYES (LYSOCHROMES) Not real dyes, lack auxochrome
Gives color to lipids because they are more soluble in lipid
medium of the tissues than in 70% alcohol
Sudan Black B, Sudan III, Sudan IV
STAINS AND STAINING SOLUTIONS
OIL SOLUBLE DYES (LYSOCHROMES)1. Sudan Black B
Most sensitive of the oil soluble dyes
Has 2 secondary amino groups per molecule
Slightly basic dye, non-specific staining
Greater affinity for phospholipids, neutral fats (triglycerides)
but not crystalline cholesterol, free fatty acids
Fat absorption of the dye is based on dye concentration,
temperature and physical state of the fats
Maximal dye uptake – melting point of fat Liquid or semi-liquid fats – stained
Crystalline or solid – not stained
STAINS AND STAINING SOLUTIONS
OIL SOLUBLE DYES (LYSOCHROMES)2. Sudan IV
Has no secondary amino group
Stains neutral fats (triglycerides) but not phospholipids or fine
lipid droplets
Benzoic acid – intensifies fat staining and prevents rapid
deterioration of the solution
Deep, intense red color
STAINS AND STAINING SOLUTIONS
OIL SOLUBLE DYES (LYSOCHROMES)3. Sudan III
Stains CNS tissues
Less deep, light orange stain
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STAINS AND STAINING SOLUTIONS
CHIEF SOLVENTS USED FOR STAINS1. Water
Distilled, unless otherwise specified
2. Alcohol
Ethyl alcohol
Methyl alcohol Absolute
Acetone-free if for use on blood stains
3. Aniline Water
10ml of aniline per ½ to 1L of hot dist. water
4. Phenol
Used in aqueous solution of 0.5 to 5%
You wish!!!
I’m not done!!!
CARBOHYDRATES
Periodic Acid Schiff
Periodic acid oxidizes 1,2 glycol group of
polysaccharides and mucin, liberating
aldehydes
Oxidation carried out with RT of 25C below.
Intensity of PAS proportional to sugar content
Schiff reagent
Basic fuschin ( rosanilin, pararosanilin, Magenta
II)
Converted to colorless leukofuschin by sulfur
oxide
Reoxidation restores magenta color
Principle: sulfuration to rearrange chromophore
group
Barger and de lamater: thionyl chloride
De tomasi-Coleman: potassium metabisulfite
Itikawa and Oguru: sulfur dioxide gas
Mucoprotein: most common PAS positive
substance
Glycogen:
Thin sections placed in 4C
PAS with Diastase:
Diastase can digest glycogen
Method of choice for glycogen staining
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Other methods:
Best carmine: selective and highly specific
• Glycogen and carmine yields bright red color
• Potassium carbonate and KCl can be added to reduce
background staining
Langhans Iodine: oldest stain and obsolete
• May be used with diastase
Mucin
Polysaccharides serving as ground substance
for connective tissue
Precipitated by dilute acetic acid, dissolved by
alkali
Classification:
Mucopolysaccharides (acid)
Mucoproteins (Neutral)
Acid Mucopolysaccharides
Polysaccharides with hexuronic acid
bound to sulfuric acid esters and proteins
Ground substance found throughout the
body
PAS negative
Alcian blue
Forms bonds with carboxyl or sulfate groups
Most popular method for acid mucins
Can be combined with PAS to stain for neutral
mucin
Acid mucin blue
Neutral mucin: magenta
Mixture: blue to purple
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Mucicarmine
Carmine with aluminum hydroxide to improve
mucin staining
Alum salts forms chelate compounds with carmine
binding to mucin containing tissue
Large molecular complex allows dinding with
acid mucins but not other acidic substances
Useful for staining encapsulated fungi
Other stains for mucin:
Acridine Orange
Orange fluorescence
Colloidal iron
Colloidal iron is adsorbed into mucin at low ph,
subsequently staining with prussian blue
Greater sensitivity than alcian blue, but more
complex and time consuming
Fats or Lipids
Best demonstrated on cryostat sections of
fresh tissues
Sudan dyes
Oil red O
Stains neutral fats and lipofuschin
• Fat: brilliant red
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CONNECTIVE TISSUE
RETICULIN (RETICULUM)
CONNECTIVE TISSUE FIBERS
Is a fibrillary extracellular matrix
Not visible on Hematoxylin-Eosin staining method
Van Gieson’s stain Unstained or faint pinkish color
Periodic Acid Schiff (PAS) Stains purplish red
Not satisfactory stain due to the delicate nature of the reticulin fibers
Silver impregnation technique Best technique because reticulin fibers are argyrophylic
Prepared by producing a precipitate from silver nitrate with sodium, potassium, or ammonium hydroxide or with lithium or sodium carbonate Reduced to silver oxide on the fibers, and reduced to black metallic silver by
formalin
Toning with yellow gold chloride gives a very pale gray background which improves subsequent counterstaining
RETICULIN (RETICULUM)
CONNECTIVE TISSUE FIBERS
Gomori’s Silver Impregnation Stain for Reticulin
NOTES:
• Ammoniacal silver solutions must be fresh
• Old silver compounds are explosive
• Glassware should be washed with nitric acid and rinsed
in distilled water
• Forceps should be nonmetallic
• Use glass-distilled water
• Mount the paraffin sections well
• Inactivate any unused solutions by adding excess of
sodium chloride solutions or of dilute hydrochloric acid
RETICULIN (RETICULUM)
CONNECTIVE TISSUE FIBERSCOLLAGEN
Forms a coarser extracellular framework than reticulin
Collagens may be differentially stained by:
1. Van Gieson’s stain
2. Masson’s Trichrome stain
3. Mallory’s Aniline Blue syain
4. Azocarmine stain
5. Krajian’s Aniline Blue stain
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COLLAGEN
Silver impregnation
Stain yellow, lavender or brown
Acid aniline dyes (aniline blue, acid fuchsin, methyl blue
or indigo carmine) from fairly strong acid solutions
Fibers stain selectively
Most commonly used acid is picric acid
COLLAGEN
Van Gieson’s stain
Stain red
Simplest method using picric acid and acid fuchsin
Nuclei Brownish black to black
Collagen (fibrous CT) Pink, or deep red
Muscle, cytoplasm, RBC, fibrin Yellow
COLLAGEN
Masson’s Trichrome Stain Uses dyes in acid solution involving nuclear staining with
iron hematoxylin, followed by cytoplasmic staining with a red dye (e.g. Ponceau phosphotungstic acid, phosphomolydicacid or both, and fixed staining of fibers with a blue or green stain (e.g. aniline blue or light green)
Fixation: Zenker, Helly, Boulin’s and Formol sublimate solutions
Sections: use paraffin sections
Muscle, RBC, and keratin Red
Nuclei Blue-black
Collagen and mucus Blue
COLLAGEN
Mallory’s Aniline Blue Stain
Not absolutely differential because it also stains hyalin fibrils,
fibroglia fibrils, smooth and striated muscle fibers and amyloid
Collagen fibers stain red
Elastic fibers stain pale pink or yellow
* If it is desired to bring out collagen fibers sharply, omit the staining
with acid fuchsin
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COLLAGEN
Azocarmine Stain
Heidenhain’s modification of Malloy’s aniline blue stain
Valuable stain showing minute details
Amyloid CT and mucus colloid Deep blue stain
Nuclei Red
ELASTIC FIBERS
Present in skin, ligaments, aorta, arterial elastic lamina, and lung
Staining methods for elastic fibers:
1. Weigert’s Elastic Tissue stain
2. Taenzer-Unna Orcein method
3. Verhoeff’s stain
4. Gomori’s Aldehyde-Fuchsin stain
5. Krajian’s method
ELASTIC FIBERS
Weigert’s Elastic Tissue stain Tissue is placed in Weigert’s stain, made up of fuchsin,
resorcin and ferric chloride, differentiated with acid-alcohol, and counterstained with neutral red, H & E, or hematoxylin and
Van Gieson’s stain.
Fixation: formalin or alcohol
Sections: thin paraffin sections
Elastic fibers appear dark-blue or blue-black on clear background.
ELASTIC FIBERS
Verhoeff’s elastic method Fixation: formalin
Section: paraffin
Elastic fiber Black
Krajian’s Technique Rapid method
Elastic fibers Bright red
Fibrin and CT Dark blue
RBC Orange-yellow
ELASTIC FIBERS
Orcein (Taenzer-Unna Orcein Method)
Vegetable dye
Demonstrate finest and most delicate fibers in skin
Differentiated with acid-alcohol and counterstained with
methylene blue or alum hematoxylin.
Elastic fibers stain dark-brown
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PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
1. Fibrin
Insoluble fibrillar protein
Forms bundles which contract into dense homogenous
masses
Seen after tissue damage, blood clots, acute inflammatory
reactions
MSB Technique (Lendrum’s Martius, Scarlet, Blue)
Employs Martius yellow, Brilliant crystal scarlet, and Soluble blue
Fibrin stains red (early fibrin may stain yellow, and very old fibrin
may stain blue)
PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
1. Fibrin
Mallory’s Phosphotungstic Acid Hematoxylin (PTAH) Method
PTAH solution, chemically oxidized with potassium permanganate
May take some months to ripen, but remain usable for many years
Fibrin stains dark blue
Note:
Omit acid dichromate treatment if tissue was fixed with chromate-
containing fixative
Dehydration must be rapid
2. Fibrinoid
• An eosinophilic material
• Identical staining reactions to fibrin
• Found in collagen diseases, hypersensitivity, SLE, rheumatic heart
diseases, in vessel walls (“necrotizing vasculitis”), and sometimes, plugs
in capillaries
3. Hyalin
• Wide variety of pathologic exudates and deposits
• Degenerated collagen, hypertension, atheroma, diabetic kidney
• Non-specifically demonstrated using Periodic Acid Schiff
PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
4. Amyloid
• Semi-translucent, ground glass or hyaline eosinophilic
substance
• Deposited in CT cells, kidney, spleen, adrenals, lymph nodes,
pancreas. TB, leprosy or osteomyelitis
• Idiopathic or primary amyloidosis in heart, tongue, larynx,
intestine, skeletal muscle and blood vessels
• Fixative: formalin (not prolonged)
PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
4. Amyloid
Methods for Amyloid Demonstration:
1. Gram’s Iodine stain
2. Congo Red method
3. Metachromatic staining
4. Induced fluorescence staining with Thioflavine
PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
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4. Amyloid
• Gram’s Iodine Stain
Amyloid appears as a delicate purple or blue color
• Congo Red Method
Fixation: not critical; formalin is satisfactory
Section: paraffin or frozen sections
Amyloid Red
Nuclei Blue
PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
4. Amyloid
• Metachromatic Staining
Methyl violet-crystal violet method
Fixation: not critical; carnoy, formol-sublimate
Section: paraffin or cryostat
Amyloid Purplish red
Nuclei Shades of violet
PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
4. Amyloid
• Induced Fluorescent Staining with Thioflavine-T
Fixation: not critical
Using UV light source, will exhibit silver-blue fluorescence
Using blue light fluorescence quartz-iodine or mercury vapor
lamp , amyloid and elastic tissue will exhibit a yellow
fluorescence
PATHOLOGIC CHANGES AND DEPOSITS
FOUND IN CONNECTIVE TISSUES
End of Presentation
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