11 staining

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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)

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


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)


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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


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


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


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.


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


NATURAL DYES

Derived from plants and animals

E.g. Hematoxylin, Cochineal dyes, Orcein,

Saffron

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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


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


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


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


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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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



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



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


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



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



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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COMMON STAINING SOLUTIONS 2.

Iron Hematoxylin

Used for regressive staining

Blue black lakes

Iron is an active oxidizing agent


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


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


COMMON STAINING SOLUTIONS 3. Eosin

3 forms:

Eosin B, Erythrosin B

Deeper red color

Eosin S, Eosin alcohol-soluble

Ethyl eosin


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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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


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


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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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


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


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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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


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


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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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


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


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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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


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


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


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


OIL SOLUBLE DYES (LYSOCHROMES)3. Sudan III

Stains CNS tissues

Less deep, light orange stain

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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


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


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)



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



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)



4. Amyloid

Methods for Amyloid Demonstration:

1. Gram’s Iodine stain

2. Congo Red method

3. Metachromatic staining

4. Induced fluorescence staining with Thioflavine



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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



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



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

11 staining

Documents

staining solutionsnatural

staining solutionsother

van giesons

periodic acid

gold chloride

elastic fibers

janus green

aluminum chloride