HISTOLOGY

LECTURE # 24

INTRODUCTION TO SPECIAL STAINS TECHNIQUES:

CARBOHYDRATES & AMYLOIDS

Rationale: Special stain techniques are one of the major processes done in the histology
laboratory. These techniques are performed to be evaluated with the diagnostics slides from
H&E’s. These stains are used as an aid and a diagnostic tool for a final diagnosis.

Objective:
Once completed this lecture, the student should be able to:

a) Describe the different types of mucosubstances.

b) Learn the various methods of stain demonstration.

c) Learn the classification for carbohydrates & Amyloid staining.

d) Learn the procedures and diagnostic tools for each stain.

CARBOHYDRATES

Carbohydrates have the job of providing all the cells in the body with the energy they need.
When carbohydrates are consumed, the body turns them into glucose, which provides
sufficient energy for everyday tasks and physical activity. If the body produces too much
glucose, it will be stored in the liver and muscle cells as glycogen, to be used for when the
body needs an extra burst of energy. Any leftover glycogen that isn't stored in liver and
muscle cells is turned into fat.

These carbohydrates include organic compounds such as sugars, starch, cellulose and polymers
that are mostly linked to proteins.

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Chemically they are ketones or aldehydes derivatives of polyhydroxy alcohols, and they may be
classified as:
Monosaccharides = one sugar unit
Oligosaccharides = few sugar units (2-10)
Polysacharides = many sugar units

Glucose
Is the only monosaccharide found in the human body and in any demonstrable quantity. It is
extremely soluble in aqueous solutions, and is small in molecular size.
It cannot be demonstrated in tissue sections.

Glycogen is a form of polymer in which carbohydrates is stored in humans. The primary

sources for the storage of glycogen are the liver and the skeletal muscle. Glycogen is broken
down into glucose to meet the energy needs of the body.

The enzymatic breakdown can occur after death, a prompt fixation is important when glycogen
demonstration is required. It is relatively insoluble in aqueous solutions and contrary to glucose
it can be demonstrated in tissue section.

Other Carbohydrates
They are either conjugated into lipid or proteins. The histochemistry is complex and confusing
because of terminology and classifications are inconsistent.

They are divided into a four group system used by Cullings.

Group I: Neutral polysaccharides

a) Glucose-containing: glycogen, starch & cellulose
b) N-Acetyl-glucosamine-containing: chitin
c) Very positive with PAS reaction
d) Negative reaction with Alcian Blue, Colloidal Iron, Mucicarmine.

Group II: Acid Mucopolysaccharides

A. Carboxylated (COOH): hyaluronic acid
1. Found in Connective tissue and umbilical cord
B. Sulfated (OSO3H) and Carboxylated (COOH):
1. Chondroitin sulfate A (chondroitin-4-sulfate)
2. Chondroitin sulfate C (chondroitin-6-sulfate)
a) Found in cartilage, chondrosarcomas, cornea & blood vessels
C. Chondroitin Sulfate B (dermatan sulfate)
1. Found in skin, connective tissue, aorta & lung.
D. Heparin
1. Found in mast cells and the intima of arteries.
E. Sulfated only (COOH-free): human aorta & bovine cornea
1. All polysaccharides in this group are acidic and thought to be attached to
protein
2. The acid mucopolysaccharides are also called connective tissue mucins
3. PAS – Negative

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Group III: Glycoproteins
a) Mucin, mucoid, mucoproteins, mucosubstances
b) Mostly “epithelial mucins”, but can occur in connective tissue.
c) Potentially, but not necessarily, PAS-positive
d) Neutral: ovimucoid (egg white), mucin in stomach, Paneth cells granules.
e) Carboxylated (COOH): sialoglycoproteins that contains sialic acid and no
sulfate.
A. Sialomucin – found in submaxillary glands mucins, small intestine mucins, fetal
mucins, upper part of colonic crypts, and human sublingual glands.
B. Sulfated only (COOH-free): human aorta & bovine cornea
C. Serum glycoproteins.
1. Blood group substances.
D. Sulfated (OSO3H) and Carboxylated (COOH): sialoglycoproteins that contains
both sialic acid & sulfate
1. Found in colonic mucins of sheeps & humans.

Group IV: Glycolipids

A. Cerebrosides: fatty residues bound to carboxylated structure.
B. Phosphatides: PAS-positive, non-carbohydrate-containing lipids, including
lecithin, cephalin, and sphingomyelin.
Polysaccharides
This occurs in the body as a mixture. A long-chain of carbohydrates polymers attached to a small
protein core, and short-chain carbohydrates polymers attached to large protein core, is not
possible.

PAS Reaction (Periodic Acid Schiff’s)

I. Purpose of the stain: To demonstrate polysaccharides, neutral mucosubstances and
basement membranes.

II. Principle of the stain: This reaction is based on oxidation of certain tissue elements to
aldehyde by use of a periodic acid solution. The most common reactive group is the 1,2
glycol group, but other groups are also selectivity oxidized by periodate. A Schiff’s
reagent is used for the detection of the glycogen, followed by a sulfurous wash and a
counterstain, such as hematoxylin or light green when demonstrating fungus.

III. Fixatives: 10% Neutral buffered formalin, Bouin’s Solution. Blood smears should be
fixed in methyl alcohol 5 to 10 minutes.

IV. Sectioning: Biopsies kidney & liver 1-2 microns. Surgical specimens 4 to 5 microns.

V. Controls: For glycogen demonstration use liver, for basement membranes

demonstration use kidney.

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 VI. Reagents:
1. 0.5% Periodic Acid: Periodic acid & Distilled water.
2. Schiff’s Reagent: Basic Fuchsin, Distilled water Sodium Metabisulfite, 1N
Hydrochloric Acid.
3. 0.55% Potassium Metabisulfite: Potassium Metabisulfite & Distilled water.
4. Hematoxylin or 0.02% Fast Green Solution: Fast Green Dye, Distilled water &
Acetic acid.
VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place sections in 0.5% Periodic Acid solution for 5 minutes. This step is where we
are changes the 1,2 glycol group to aldehyde groups.
3. Wash Slides in three changes of distilled water. This step is necessary for the rinse
of the Periodic Acid.
4. Place sections in Schiff’s reagent for 15 minutes. This step is where we are staining
the section for glycogen demonstration.
5. Wash slides for 1 minute in each of two coplin jars of 0.55% potassium
Metabisulfite. This step is to remove the excess non specific staining.
6. Wash slides in running tap water for 10 minutes. This step is in place in order to
develop the full color of the structure or substance in question.
7. Counterstain slides for 30 seconds in a Harris’ hematoxylin solution containing
acetic acid (2 ml acetic acid to 48 ml of Hematoxylin) or Fast green Solution
containing acetic acid (1 drop of acetic acid for every 60 ml of Fast Green solution).
This step will create a background contrast within the substance stain and the tissue.
8. Wash slides well in tap water to blue the hematoxylin. If Fast green is used
continue with the dehydration process.
9. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip with
a resinous mounting media.
Hematoxylin

Schiff’s 0.55%
Sodium
.02% Light Green

Reaction
Metabisulfit
e
Dehydration
Dehydration

Water Water
Rinses Rinses

Testing the Schiffs Reagent:

1. Place 10 ml of 37 – 40% Formaldehyde in a beaker.
2. Add a few drops of the Schiff’s reagent into the beaker.
3. If the solution turns immediately to a reddish purple, it is still good.
4. If the solution delays in turning and the results are of a deep blue-purple color. The solution
is breaking down and needs to be replaced.

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VIII. Results:
Glycogen ……………………………….. Bright Rose
Neutral mucosubstances ……………….. Bright Rose
Epithelial substances …………………… Bright Rose
Basement Membrane …………………… Bright Rose
Fungal Walls …………………………… Bright Rose
Nuclei/Background with Hematoxylin..... Blue
Background with Fast Green ……………Green

Glycogen in Liver Neutral Mucosubstances in Colon

Demonstration of Fungus Demonstration of Basement Membrane

(Polysaccharide) in kidney (Glomeruli)

PAS Reaction With Diastase Digestion

I. Purpose of the stain: To demonstrate glycogen in tissue sections.

II. Principle of the stain: This is a sensitive histochemical method for glycogen. Diastase
and α-amylase act on glycogen to depolymerize it into smaller sugar units (maltose and
glucose) that are washed out of the section.

III. Fixatives: 10% Neutral buffered formalin, formalin alcohol, or absolute alcohol.

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IV. Sectioning: Biopsies kidney & liver 1-2 microns. Surgical specimens 4 to 5 microns.
Need to cut two slides and label one slide “with” and the other slide “without”.

V. Controls: Two control sections of liver containing glycogen, one labeled “with” and
the other labeled “without”.

VI. Reagents:
1. 0.5% Periodic Acid: Periodic acid & Distilled water.
2. Schiff’s Reagent: Basic Fuchsin, Distilled water Sodium Metabisulfite,
1N Hydrochloric Acid.
3. 0.55% Potassium Metabisulfite: Potassium Metabisulfite & Distilled water.
4. Hematoxylin or 0.02% Fast Green Solution: Fast Green Dye, Distilled water &
Acetic acid.
5. Malt Diastase Solution: Diastase of Malt & Phosphate buffer, pH 6.0
6. Phosphate buffer, pH 6.0: Sodium Chloride, Sodium Phosphate (Monobasic), &
distilled water. pH if necessary.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides labeled “with” in preheated diastase solution at 37oC for 1 hour.
3. Hold slides labeled “without” in distilled water. This step is depolymerizing the
glycogen in the tissue section.
4. Wash slides in distilled water to remove the diastase solution of the tissue
sections.
5. Place all slides “with” & “without” in 0.5% Periodic Acid solution for 5 minutes.
This step is where we are changes the 1,2 glycol group to aldehyde groups.
6. Wash Slides in three changes of distilled water. This step is necessary for the
rinse of the Periodic Acid.
7. Place sections in Schiff’s reagent for 15 minutes. This step is where we are
staining the section for glycogen demonstration.
8. Wash slides for 1 minute in each of two coplin jars of 0.55% potassium
Metabisulfite. This step is to remove the excess non specific staining.
9. Wash slides in running tap water for 10 minutes. This step is in place in order to
develop the full color of the structure or substance in question.
10. Counterstain slides for 30 seconds in a Harris’ hematoxylin solution containing
acetic acid (2 ml acetic acid to 48 ml of Hematoxylin).
11. Wash slides well in tap water to blue the hematoxylin.
12. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

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 “With” “With” “With” “With” “With”

Hematoxylin
Schiff’s 0.55%
Reaction Sodium
Metabisulfit
e

------------

Dehydration
Water Water
Diastase 0.5% Rinses Rinses “Without”
Digestion Periodic
Acid

Notes:
™ Saliva can be used for 20 minutes at room temperature.
™ Disadvantage:
1. If person suffers from amylase deficiency, then the
procedure will not work.
2. Mycobacterium can also make their way on to the tissue
section.
™ Glycogen sections fixed in picric-acid containing fixatives may
show some resistance to diastase digestion.

VIII. Results:
Slides labeled “without”
Glycogen ……………………………….. Bright Rose
Nuclei/Background with Hematoxylin..... Blue

Slides labeled “with”

Glycogen ……………………………….. Absent/No color
Nuclei/Background with Hematoxylin..... Blue

No Digestion Digestion Digestion

Glycogen No Glycogen No Glycogen
presence of leucofuchsin

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Mayer’s Mucicarmine

I. Purpose of the stain: Staining of “Epithelial” mucin in tissue sections.

II. Principle of the stain: This is an empirical stain. Aluminum is believed to form a
chelating complex with carmine; the resulting compound has a net positive (+) charge
and attaches to the acid groups of mucin.

III. Fixatives: 10% Neutral buffered formalin.

IV. Sectioning: Cut Paraffin sections from 4 to 5 microns.

V. Controls: Colon, Small intestine, or Appendix.

VI. Reagents:
1. Mucicarmine Stock Solution: Carmine, Alum lake, Aluminum hydroxide, 50%
ethyl Alcohol, 25% ethyl alcohol, aluminum chloride, anhydrous.
2. Mucicarmine Working Solution: Mucicarmine Stock Solution & Distilled water.
Prepare just before use.
3. Weigert’s Iron Hematoxylin:
Solution A: Hematoxylin & 95% Alcohol
Solution B: Hydrochloric Acid, concentrate, 29% Ferric Chloride solution
& Distilled water.
4. 0.25% Metanil Yellow Solution: Metanil Yellow, Distilled water & Glacial
Acetic acid.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place sections in Working Iron Hematoxylin, (by mixing equal parts of Solution
A & Solution B) for 7 minutes. This step will be staining the nuclear details
(Nuclei).
3. Wash Slides in running water for 10 minutes. This step is necessary in order to
remove any excess Iron Hematoxylin.
4. Place sections in Working Mucicarmine for 1 hour. This step is where the mucin
substances will be staining.
5. Rinse slide quickly in distilled wash and remove any excess water before going
into the next step. This step is to remove the excess non specific staining.
6. Stain slides in Metanil Yellow Solution for 30 seconds to 1 minute. This step will
create a background contrast within the substance stained and the tissue.
7. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

8
 Mucin Metanil
Solution Yellow

Dehydration
Water Water
Wiegert’s Rinses Rinses
Hematoxylin Mayer’s Mucicarmine Stain in Colon

VIII. Results:
Mucin …………………………….……...Deep rose to Red
Capsules of Cryptococcus ……………….Deep rose to Red
Nuclei…………………………………….Black
Background/Other Tissue Elements……...Yellow

Alcian Blue, pH 2.5

I. Purpose of the stain: Demonstration of acid mucopolysaccharides.

II. Principle of the stain: Alcian Blue is a basic dye containing copper which gives it
colored. When used in a 3% acetic acid solution (pH 2.5), Alcian blue stains for
sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated
sialomucins (glycoproteins). It is believed to form a salt link with the acid groups of
acid mucopolysaccharides.

III. Fixatives: 10% Neutral buffered formalin or Bouin’s solution.

IV. Sectioning: Cut Paraffin sections from 4 to 5 microns.

V. Controls: Colon, Small intestine, or Appendix.

VI. Reagents:
1. 3% Acetic acid Solution: Glacial acetic acid & Distilled water.
2. 1% Alcian Blue Solution: Alcian Blue 8GX dye, 3% Acetic acid solution & few
crystal of thymol.
3. Nuclear Fast Red: Nuclear-fast Red dye, Aluminum sulfate, Distilled water &
grains of thymol.

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 VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place sections in 3% Acetic acid solution for 3 minutes. This step is necessary in
order to prepare the tissue to stain for sulfated and carboxylated acid
mucopolysaccharides.
3. Place slides in Alcian Blue solution for 30 minutes. This step will allow the acid
mucopolysaccharides staining.
4. Wash Slides in running water for 10 minutes. This step is necessary in order to
remove any excess Alcian Blue Solution.
5. Rinse slides in distilled water.
6. Stain slides in Nuclear Fast Red Solution for 5 minutes. This step will create a
background contrast within the substance stained and the tissue.
7. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

NFR

Water Water
3% Acetic Alcian Blue Rinses Rinses
Acid pH 2.5 Acid mucopolysaccharides in Colon

VIII. Results:
Weakly acid sulfated mucosubstances, hyaluronic acid and sialomucin .……..Dark Blue
Background …………………………………………………………………... Pink to red

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Alcian Blue, pH 1.0
I. Purpose of the stain: Demonstration of sulfated mucosubstances.

II. Principle of the stain: When used in a 0.1N hydrochloric acid solution (pH 1.0),
Alcian blue stains only sulfated acid mucopolysaccharides and sulfated sialomucins
(glycoproteins). Acid mucopolysaccharides that are carboxylated only will not stain.

III. Fixatives: 10% Neutral buffered formalin or Bouin’s solution.

IV. Sectioning: Cut Paraffin sections from 4 to 5 microns.

V. Controls: Colon, Small intestine, or Appendix.

VI. Reagents:
1. 0.1N Hydrochloric acid solution: Concentrated hydrochloric acid & Distilled
water. Never add water to acid.
2. 1% Alcian Blue Solution: Alcian Blue 8GX dye, 0.1N Hydrochloric acid.
3. Nuclear Fast Red: Nuclear-fast Red dye, Aluminum sulfate, Distilled water &
grains of thymol.
VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in 1% Alcian Blue in 0.1N Hydrochloric acid solution for 30
minutes. This step will allow the sulfated mucosubstances.
3. Rinse slides briefly in 0.1N Hydrochloric acid.
4. Blot slides dry with fine filter paper. Do not wash with water since this may
change the pH and cause non specific staining.
5. Stain slides in Nuclear Fast Red Solution for 5 minutes. This step will create a
background contrast within the substance stained and the tissue.
6. Wash slides in distilled water.
7. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and
coverslip with a resinous mounting media.

NFR
Blot the Section

0.1N Water
Alcian Blue HCl Rinses
pH 1.0
Sulfated mucosubstances in Colon

VIII. Results:
Sulfated mucosubstances..…………………….……...Pale Blue
Background …………………………………... Pink to red

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Mueller-Maury Colloidal Iron
I. Purpose of the stain: Demonstration of carboxylated & sulfated mucopolysaccharides.

II. Principle of the stain: Colloidal ferric iron is, at low pH, absorbed principally by
carboxylated & sulfated mucosubstances. The excess reagent is washed out, and
Prussian blue will detect the iron bound to the tissue.

III. Fixatives: 10% Neutral buffered formalin, Carnoy’s & alcoholic formalin’s.

IV. Sectioning: Cut Paraffin sections from 4 to 5 microns.

V. Controls: Colon, Small intestine, or Appendix.

VI. Reagents:
1. Mueller Colloidal Iron Stock: 29% Ferric chloride & distilled water.
2. Working Colloidal Iron: Stock solution, distilled water & acetic acid.
3. 2% Potassium ferrocyanide: Potassium ferrocyanide & Distilled water
4. 2% hydrochloric acid: Hydrochloric acid & distilled water
5. Ferrocyanide-Hydrochloric acid: Equal mix of 2% Potassium ferrocyanide &
2% hydrochloric acid.
6. 12% Acetic acid: Acetic acid & distilled water.
7. Nuclear Fast Red: Nuclear-fast Red dye, Aluminum sulfate, Distilled water &
grains of thymol.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in 12% Acetic acid solution. This step prevents watery dilution of
the colloidal iron.
3. Place slides in Working Colloidal iron prepared fresh for 1 hour. This step is
bounding the ferric ions to the tissue.
4. Rinse slides in three changes of acetic acid for 3 minutes each change.
5. Immerse slides in ferrocyanide-hydrochloric solution for 20 minutes at room
temperature. This step will bound the dye to the ferric ions on the tissue.
6. Wash slides in running water. This will remove any excess ferric solution.
7. Stain slides in Nuclear Fast Red Solution for 5 minutes. This step will create a
background contrast within the substance stained and the tissue.
8. Wash slides in running tap water for at least 1 minute.
9. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

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 NFR

12% Acetic acid changes

Working Colloidal Iron

Ferrocyanide-hydrochloric soln
Water
12% Acetic Rinses
acid

VIII. Results:
Acid mucopolysaccharides & sialomucins ……...Deep Blue
Nuclei ……………………………………………Pink-red
Cytoplasm ………………………….…………... Pink

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Alcian Blue with Hyaluronidase
I. Purpose of the stain: Differentiate epithelial and connective tissue mucins.

II. Principle of the stain: Staining will disappear or be dramatically reduced when tissue
sections containing hyaluronic acid, chondroitin sulfate A, or chondroitin sulfate C
(“connective tissue” mucin) are digested with testicular hyaluronidase. Glycoproteins
(“epithelial” mucins) will not be affected.

III. Fixatives: 10% Neutral buffered formalin is preferred.

IV. Sectioning: Cut two paraffin sections from 4 to 5 microns. Label one “without
digestion” and the other “with digestion.”
V. Controls: Two sections of umbilical cord should be used and labeled as “with
digestion” and “without digestion.”

VI. Reagents:
1. 0.1M Potassium Phosphate, Monobasic: Potassium phosphate & distilled water.
2. 0.1M Sodium Phosphate, dibasic: Sodium Phosphate & distilled water.
3. Buffer Solution: 0.1M Potassium phosphate & 0.1M Sodium phosphate.
4. Hyaluronidase Digestion Solution: Testicular hyaluronidase & buffer solution,
mixed before use.
5. Alcian Blue staining solution: Alcian Blue 8GX & 3% Acetic acid.
6. Nuclear Fast Red: Nuclear-fast Red dye, Aluminum sulfate, Distilled water &
grains of thymol.

VII. Procedure:
1. Prepare buffer and digestion solutions. Preheat one coplin jar of each at 37oC
for 1 hour.
2. Deparaffinize and hydrate slides to water.
3. Place slides labeled “with digestion” in the digestion solution, and the slides
labeled “without digestion” in the buffer solution. Incubate at 37oC for 2 hours.
This step is creating the digestion process.
4. Wash both sets of slides in running tap water for 5 minutes. (slides can be
combined at this point).
5. Place slides in 3% Acetic acid solution for 3 minutes.
6. Place slides in Alcian blue, pH 2.5 solution for 30 minutes.
7. Wash slides in running tap water for 10 minutes.
8. Rinse slides in distilled water
9. Counterstain slides in Nuclear fast red solution for 5 minutes.
10. Wash slides in running tap water for at least 1 minute.
11. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

14
 Nuclear Fast Red
“With” “With” “With” “With”
Alcian Blue,
pH 2.5

Water Rinses
ate
r
Ri
ns
es ------------

Dehydration
Water
Hyaluronidase 3% Acetic acid Rinses “Without”
Digestion

VIII. Results:
Sections “without digestion, Acid mucopolysaccharides & sialomucins …………….Deep Blue
In the sections “with digestion” mucosubstances containing hyaluronic acid and chondroitin
sulfates A & C ………………………………………………..………… Marked loss of staining.

“Without Digestion” “With Digestion”

Acid mucopolysaccharides & sialomucins Loss of mucosubstances containing
hyaluronic acid

15
Alcian Blue/PAS
I. Purpose of the stain: Differentiate between neutral and acidic mucosubstances.

II. Principle of the stain: Staining for the acidic mucosubstances will be done with the
Alcian Blue technique and the neutral mucosubstances by the PAS reaction.

III. Fixatives: 10% Neutral buffered formalin or Zenker’s solution.

IV. Sectioning: Cut paraffin sections from 4 to 5 microns. Kidney & liver biopsies from 2
to 3 microns.

V. Controls: Use kidney or mucin control, depending on the diagnostic tissue to be

stained.

VI. Reagents:
1. 3% Acetic acid Solution: Glacial acetic acid & Distilled water.
2. 1% Alcian Blue Solution: Alcian Blue 8GX dye, 3% Acetic acid solution &
few crystal of thymol.
3. 0.5% Periodic Acid: Periodic acid & Distilled water.
4. Schiff’s Reagent: Basic Fuchsin, Distilled water Sodium Metabisulfite,
1N Hydrochloric Acid.
5. Stock Reducing Rinse: sodium Metabisulfite & Distilled water.
6. Working Reducing Rinse: Stock Reducing Rinse & Distilled water.
7. Harris’ Hematoxylin

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in 3% acetic acid for 1 minute.
3. Place slides in Alcian blue solution for 30 minutes.
4. Wash sections in running tap water, then rinse slides in distilled water.
5. Place sections in 0.5% Periodic Acid solution for 10 minutes. This step is
where we are changes the 1,2 glycol group to aldehyde groups.
6. Wash Slides in three changes of distilled water. This step is necessary for the
rinse of the Periodic Acid.
7. Place sections in Schiff’s reagent for 15 minutes. This step is where we are
staining the section for glycogen demonstration.
8. Wash sections in running tap water for 5 minutes, then rinse slides in distilled
water.
9. Place slides in reducing rinse solution for 5 minutes.
10.Wash slides in running tap water for 10 minutes.
11.Stain sections in Harris’ Hematoxylin with acetic acid for 1 minute. This step is
optional.
12.Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

16
 Nu
cle
“With” “With” “With” ar “With”
Alcian Blue,
Fa
pH 2.5
st
Re
W W d
ate ate
r r
Ri Ri
ns ns
es es De ------------
hy
dr
ati
on

Water
Hyaluronidase 3% Acetic acid Rinses “Without”
Digestion

VIII. Results:
Exclusively Acid mucopolysaccharides...............................................Blue
Neutral Polysaccharides.................................................................Magenta
Certain substances will be colored by both PAS & Alcian blue.......Purple

AMYLOIDS

" A fibrillar protein that deposits in tissue under certain pathologic conditions.

" Contains 1-2% carbohydrate, mostly acid mucopolysaccharides.

" Amyloidosis is a disease characterized by an amorphous, eosinophilic, extracellular

deposit that gradually replaces cellular elements of vital organs and causes progressive
loss of function and eventual death.

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 Amyloid is classified into four groups:
I. Primary Amyloid - occurs spontaneously in the absence of any predisposing
disease.
" Organ most affected: muscle, heart, skin & tongue.

II. Secondary Amyloid - Inflammatory such as rheumatoid arthritis and tuberculosis.

" Organ most affected kidneys, liver, spleen & adrenal glands.

III. Myeloma-associated Amyloid associated with disease of immunological system and

resembles primary Amyloid in distribution.

IV. Tumor associated Amyloid.

" Found in tumors

Alkaline Congo Red Method

I. Purpose of the stain: Demonstration of Amyloid in tissue.

II. Principle of the stain: Green birefringence is the most specific technique for this
demonstration. Congo red is a Benzedrine derivative that can react with cellulose, and
Amyloid resembles cellulose in its chemical reactions. Is a linear molecule, which
allows azo and amine groups of the dye to form hydrogen bonds with similar
hydroxyl radicals of the Amyloid.

III. Fixatives: Alcohol or Carnoy’s solution is preferred. 10% NBF, Bouin’s or Zenker’s
may be used.

IV. Sectioning: Cut paraffin sections from 6 to 10 microns. If thinner may not show the
green birefringence.

V. Controls: Sections containing Amyloid may be used. It’s better not to keep many
controls pre-cut.

VI. Reagents:
1. Stock 80% Alcohol Saturated with Sodium Chloride: Sodium chloride &
Distilled water, then add 100% Ethyl alcohol.
2. Alkaline salt solution: Stock 80% alcohol saturated with sodium chloride, 1%
sodium hydroxide; filter and use within 15 minutes.
3. Stock Congo Red Staining solution: Congo Red dye, Stock 80% alcohol
Saturated with sodium chloride.
4. Working Congo Red Staining Solution: Stock Congo Red solution, 1% sodium
hydroxide; filter and use with 15 minutes.
5. Harris’ Hematoxylin.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain slides in Harris’ hematoxylin with acetic acid. This step will stain all the
nuclear details of the tissue.
3. Wash slides in running tap water for several minutes.
4. Place slides in alkaline salt solution for 20 minutes. Depress dye ionization and
acid-base type staining

18
 5. Stain slides in working Congo Red solution for 20 minutes. This step will start
the birefringence staining of the Amyloid.
6. Dehydrate rapidly in three changes of absolute alcohol, 5 or 6 good dips in each
change.
7. Clear in two to three changes of xylene and mount in synthetic resin.

Light
Microscopy

W
ate
r
Ri
ns
es De ------------
hy
dr
ati
on

Harris’ Alkaline Salt Congo Red Polarized

Hematoxylin Solution Solution Microscope

VIII. Results:
Amyloid ............................................... Deep pink to red
Elastic tissue .................................................... pale pink
Nuclei ..................................................................... Blue

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Crystal Violet

I. Purpose of the stain: Rapid demonstration of Amyloid, but not specific as Congo
Red.

II. Principle of the stain: This will induce only weak metachromasia with thionin and
toluidine blue and investigators have been unable to find a parallel reaction with
methyl violetin a wide range of mucopolysaccharides.

III. Fixatives: 10% NBF.

IV. Sectioning: Cut paraffin sections from 10 to 12 microns.

V. Controls: Sections containing Amyloid may be used. It’s better not to keep many
controls pre-cut.

VI. Reagents:
1. Stock Saturated Crystal Violet Solution: Crystal violet & 95% alcohol.
2. Working Crystal violet solution: Stock Crystal violet, distilled water &
hydrochloric acid.
VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain slides in working crystal violet solution for 5 minutes. Check the control
slide microscopically; stain longer if needed.
3. Rinse sections well in tap water.
4. Mount with an aqueous mounting medium.
5. Seal the edges of coverslip with fingernail polish.
Water Rinses

Crystal
Violet

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 VIII. Results:
Amyloid ............................................... Purplish violet
Other tissue elements ............................................ blue

Thioflavine T Fluorescent Method

I. Purpose of the stain: Is a good method for Amyloid, but not specific.

II. Principle of the stain: Thioflavin T is a fluorescent dye that attaches to Amyloid and
requires no differentiation. Background nuclear staining is quenched by staining with
aluminum hematoxylin.

III. Fixatives: 10% NBF.

IV. Sectioning: Cut paraffin sections from 6 to 10 microns.

V. Controls: Sections containing Amyloid may be used. It’s better not to keep many
controls pre-cut.

VI. Reagents:
1. 1% Thioflavine T Solution: Thioflavine T & distilled water.
2. 1% Acetic acid: Acetic acid & distilled water.
3. Mayer’s hematoxylin

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain slides in Mayer’s hematoxylin for 2 minutes. This step will quench the
nuclear fluorescence.
3. Wash slides in water for 3 to 5 minutes.
4. Stain slides in filtered Thioflavine T solution for 3 minutes.
5. Rinse slides in distilled water.
6. Differentiate slides in 1% acetic acid for 20 minutes.
7. Wash slides in running tap water for 2 minutes.
8. Blot slides dry.
9. Mount slides with a fluorescent Mounting medium.

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 Water Rinses

1% acetic acid
Mayer’s Thioflavine T
Hematoxylin

VIII. Results:
Amyloid fluoresces yellow to yellow-green

22