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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)

MAJOR GROUPS OF TISSUE STAINING

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. Perls 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, enzymelabeled or fluorescent-labeled)

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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

METHODS OF STAINING
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 Ehrlichs hematoxylin. Iron in Weigerts 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 Loefflers methylene blue, Phenol in Carbol thionine and Carbol fuchsin

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 STAINING
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 formalinfixed tissues.

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 Neutral red Safranin O Carmine Hematoxylin

Blue
Methylene blue Toluidine blue Celestine blue

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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

Routine Hematoxylin and Eosin (H & E)

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

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.

H and E PROCEDURE
6. Stain with Harris Alum Hematoxylin for 5 minutes (Ehrlichs 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.

5. Rinse in running water for a minute. 10. Use ammonia water (average of 5 minutes) or 1% aqueous lithium carbonate until the section appears blue (about 30 seconds)

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H and E PROCEDURE
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 RESULT
Nuclei blue to blue black Karyosome dark blue Cytoplasm pale pink RBCs, eosinophilic granules, keratin brightorange red Calcium and decalcified bone purplish blue Decalcified bone matrix, collage and osteoid pink Muscle fibers deep pink

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

STAINS AND STAINING SOLUTIONS

NATURAL DYES
1. Hematoxylin
Ripening
Natural ripening
Expose the substance to air and sunlight A slow process, 3-4 months Chemical oxidation Hydrogen peroxide, mercuric oxide, potassium permanganate, Sodium perborate, sodium iodate Excessive oxidation

Artificial ripening

Used in combination with a mordant such as alum, iron, chromium and copper salts

Over-ripening

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 Picrocarmine Neuropathological stain Bests carmine Demonstration of glycogen

With picric acid

With aluminum chloride

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)
Erlichs sodium iodate Harris mercuric chloride

Forms blue lakes

STAINS AND STAINING SOLUTIONS

COMMON STAINING SOLUTIONS
1. Hematoxylin B. Erlichs 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. Coles Hematoxylin
Ripened with alcoholic iodine

E. Mayers 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. Weigerts Hematoxylin
Standard iron hematoxylin Used in demonstrating connective tissue

B. Heidenhains 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 Ethyl eosin

STAINS AND STAINING SOLUTIONS

OTHER STAINS
1. Acid Fuchsin-Picric Acid (Van Giesons 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

Eosin S, Eosin alcohol-soluble

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STAINS AND STAINING SOLUTIONS

OTHER STAINS
3. 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 STAINS
6. Basic Fuchsin Plasma stain For staining acid-fast organisms, for mitochondria, for differentiation of smooth muscles (with the use of picric acid) Feulgens and Schiffs reagent for detection of aldehydes Van Giesons solution for staining connective tissues, mucin and elastic tissues

STAINS AND STAINING SOLUTIONS

OTHER STAINS
6. Basic Fuchsin A. Carbol-Fuchsin B. Colemans Feulgen Reagent C. Schiffs Reagent D. Mallorys Fuchsin Stain E. Aldehyde Fuchsin (Gomoris stain)

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STAINS AND STAINING SOLUTIONS

OTHER STAINS
7. Benzidine For staining hemoglobin 8. Bismarck Brown Used as counterstain for Grams technique, for acid fast, for Papanicolau method Used for staining diphtheria organisms

STAINS AND STAINING SOLUTIONS

OTHER STAINS
9. 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 STAINS
11. 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 STAINS
13. 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 STAINS
15. 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 Grams iodine
Stains microorganisms and fibrin in tissue sections

Lugols iodine
Used as test for glycogen, amyloid and corpora amylacea

STAINS AND STAINING SOLUTIONS

OTHER STAINS
16. 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 STAINS
18. 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 STAINS
20. 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 STAINS
22. Picric Acid Counterstain for acid fuchsin, connective tissues (in Van Giesons 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 STAINS
24. 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 STAINS
1. 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 to 1L of hot dist. water 4. Phenol Used in aqueous solution of 0.5 to 5%

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.

You wish!!! Im not done!!!

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

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

Principle: sulfuration to rearrange chromophore group

Barger and de lamater: thionyl chloride De tomasi-Coleman: potassium metabisulfite Itikawa and Oguru: sulfur dioxide gas

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Mucin
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

Polysaccharides serving as ground substance for connective tissue Precipitated by dilute acetic acid, dissolved by alkali Classification:
Mucopolysaccharides (acid) Mucoproteins (Neutral)

Acid Mucopolysaccharides
Alcian blue Polysaccharides with hexuronic acid bound to sulfuric acid esters and proteins Ground substance found throughout the body PAS negative
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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RETICULIN (RETICULUM) CONNECTIVE TISSUE FIBERS

Is a fibrillary extracellular matrix Not visible on Hematoxylin-Eosin staining method Van Giesons 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

CONNECTIVE TISSUE

RETICULIN (RETICULUM) CONNECTIVE TISSUE 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

Gomoris Silver Impregnation Stain for Reticulin NOTES:
Ammoniacal silver solutions must be fresh
Old silver compounds are explosive

COLLAGEN
1. 2. 3. 4. 5. Forms a coarser extracellular framework than reticulin Collagens may be differentially stained by: Van Giesons stain Massons Trichrome stain Mallorys Aniline Blue syain Azocarmine stain Krajians Aniline Blue stain

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

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COLLAGEN
Silver impregnation
Stain yellow, lavender or brown

COLLAGEN
Van Giesons stain
Stain red Simplest method using picric acid and acid fuchsin Nuclei Collagen (fibrous CT) Muscle, cytoplasm, RBC, fibrin Brownish black to black Pink, or deep red Yellow

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
Massons 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, Boulins and Formol sublimate solutions Sections: use paraffin sections Muscle, RBC, and keratin Red Nuclei Blue-black Collagen and mucus Blue

COLLAGEN
Mallorys 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
Heidenhains modification of Malloys aniline blue stain Valuable stain showing minute details Amyloid CT and mucus colloid Nuclei Deep blue stain Red

ELASTIC FIBERS
1. 2. 3. 4. 5. Present in skin, ligaments, aorta, arterial elastic lamina, and lung Staining methods for elastic fibers: Weigerts Elastic Tissue stain Taenzer-Unna Orcein method Verhoeffs stain Gomoris Aldehyde-Fuchsin stain Krajians method

ELASTIC FIBERS
Weigerts Elastic Tissue stain
Tissue is placed in Weigerts stain, made up of fuchsin, resorcin and ferric chloride, differentiated with acid-alcohol, and counterstained with neutral red, H & E, or hematoxylin and Van Giesons stain. Fixation: formalin or alcohol Sections: thin paraffin sections

Elastic fibers appear dark-blue or blue-black on clear background.

ELASTIC FIBERS
Verhoeffs elastic method
Fixation: formalin Section: paraffin Elastic fiber

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

Black

Krajians Technique
Rapid method Elastic fibers Fibrin and CT RBC Bright red Dark blue Orange-yellow

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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 (Lendrums 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

Mallorys 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 chromatecontaining fixative Dehydration must be rapid

PATHOLOGIC CHANGES AND DEPOSITS FOUND IN CONNECTIVE TISSUES

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. Grams Iodine stain 2. Congo Red method 3. Metachromatic staining 4. Induced fluorescence staining with Thioflavine

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PATHOLOGIC CHANGES AND DEPOSITS FOUND IN CONNECTIVE TISSUES

4. Amyloid Grams 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 Nuclei Red 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 Nuclei Purplish red 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

End of Presentation

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