HISTOLOGY

LECTURE # 27

INTRODUCTION TO SPECIAL STAINS TECHNIQUES:

CONNECTIVE TISSUE & MUSCLE FIBER STAINS

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 connective tissue staining.

b) Learn the various methods of stain demonstration.

c) Learn the classification for connective tissue, basement membranes & Muscle staining.

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

CONNECTIVE TISSUE

Introduction

Connective Tissues are responsible for providing and maintaining form to the body. They
function in a mechanical role, providing a matrix that connects and binds the cells and organs
and ultimately gives support to the body.

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FIBERS OF CONNECTIVE TISSUE PROPER
A. Collagen fibers

They provide strength, the more cartilage the stronger the tissue is. It is demonstrated
using the Masson & Gomori techniques and with the Van Gieson stain. The only
monosaccharide found in the human body.

B. Elastic fibers

They are abundant in tissue that needs flexibility, as they allow tissue to stretch. These
fibers cannot usually be seen in H&E stains. Special stains for these fibers are Verhoeff’s
van Gieson, Weigerts Stain, or Gomori.

C. Reticular fibers

They have identified as a type of collagen. They may be demonstrated with argyrophillic
reactions, as they have the ability to absorb silver from a solution. The silver may then
be reduced to a metallic form.

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CELLS OF CONNECTIVE TISSUE PROPER
™ Fibroblasts – most common cells in connective tissue.
™ Mesenchymal – these are primitive cells that may develop into other
differentiated cell types if the need arises for replacement.
™ Adipose or Fat - these synthesize and store lipid and are more common in most
loose connective tissue.
™ Mast cells – contains abundant secretory granules. These granules contain heparin
& histamine and exhibit metachromasia when stained with Toluidine Blue.
™ Macrophages – they are known as the “big eaters” or scavenger cells that are
found not only in connective tissue but in a variety of other tissue as liver,
myeloid, and lymphatic tissue.
™ Plasma cells – they derive from B lymphocytes produce immunoglobulins.
™ Blood cells – may be found in tissue.

Collagen

Collagen is the main protein of connective tissue. It has great tensile strength, and is the main
component of ligaments and tendons. It is responsible for skin elasticity, and its degradation
leads to wrinkles that accompany aging. Collagen also fills out the cornea where it is present in
crystalline form.

Collagen is the most abundant protein in mammals.

Collagen has an unusual amino acid composition. It contains large amounts of glycine and
proline, as well as two amino acids that are not inserted directly by ribosomes – hydroxyproline
and hydroxylysine – the former composing a rather large percentage of the total amino acids.
They are derivatised from proline and lysine in enzymatic processes of post translational
modification, for which vitamin C is required. This is related to why vitamin C deficiencies can
cause scurvy, a disease that leads to loss of teeth and easy bruising caused by a reduction in
strength of connective tissue due to, a lack of collagen, or defective collagen.

The white collagen that makes up the matrix of most connective tissue in mammals consists of
inter-woven fibers of the protein collagen. The collagen fibers consist of globular units of the
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collagen sub-unit tropocollagen. Tropocollagen sub-units spontaneously arrange themselves
under physiological conditions into staggered array structures stabilized by numerous hydrogen
and covalent bonds. Tropocollagen sub-units are left-handed triple helices where each strand is,
further, a right-handed helix itself. Thus, tropocollagen may be considered to be a coiled coil.

Another rare feature of collagen is its regular arrangement of amino acids in each of the alpha
chains of the collagen sub-units. The sequence generally follows the pattern Gly-X-Y, where Gly
for glycine, X for proline, and Y for proline or hydroxyproline. There are very few other proteins
with such regularity. The inordinate number of Gly residues allows the otherwise sterically
disallowed, tight coiling of each of the alpha chain subunits of tropocollagen, where there is a
rise per turn of just 0.3 nm as opposed to the .36 nm of a regular Alpha helical coil.
Hydroxylysine and hydroxyproline play important roles in the stabilization of the tropocollagen
globular structure as well as the final fiber shaped structure by forming covalent bonds. The
resulting structure is called a collagen helix.

Types of collagen
Collagen occurs in many places throughout the body, and occurs in different forms known as
types:

• Type I collagen - This is the most abundant collagen of the human body. It is present in
scar tissue, the end product when tissue heals by repair. It is found in tendons and the
organic part of bone.
• Type II collagen - Articular cartilage
• Type III collagen - This is the collagen of granulation tissue, and is produced quickly by
young fibroblasts before the tougher type I collagen is synthesized.
• Type IV collagen - Basal lamina
• Type V collagen - most interstitial tissue, assoc. with type I
• Type VI collagen - most interstitial tissue, assoc. with type I
• Type VII collagen - epithelia
• Type VIII collagen - some endothelial cells
• Type IX collagen - cartilage, assoc. with type II
• Type X collagen - hypertrophic and mineralizing cartilage
• Type XI collagen - cartilage
• Type XII collagen - interacts with types I and III

Muscle is a very specialized tissue that has both the ability to contract and the ability to conduct
electrical impulses. Muscles are classified both functionally as either voluntary or involuntary
and structurally as either striated or smooth. From this, there emerge three types of muscles:
smooth involuntary (smooth) muscle, striated voluntary (skeletal) muscle and striated
involuntary (cardiac) muscle. The names in the brackets are the common names given to the
particular classification of muscle.

Basement Membrane, also known as Basal Lamina, is a layer of proteins and glycoproteins that
surrounds our tissues. Cancer cells that are in the process of metastasizing must cross the basal
lamina to enter blood vessels or other areas of the body.

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Masson’s Trichrome Stain

I. Purpose of the stain: To differentiate between collagen and smooth muscle in tumors
and to identify increases in collageneous tissue in diseases such as cirrhosis of the liver.

II. Principle of the stain: This reaction is based on a three dye stain.

III. Fixatives: Bouin’s solution is preferred, but 10% Neutral buffered formalin may be
used.

IV. Sectioning: Paraffin sections at 4-5 microns.

V. Controls: Every tissue has an internal control, but uterus, small intestine, appendix or
fallopian tube will be good material.

VI. Reagents:
1. Bouin’s Solution: Picric acid saturated, formaldehyde & Glacial acetic acid.
2. Weigert’s Iron Hematoxylin: Solution A: Hematoxylin & 95% Alcohol; &
Solution B: 29% Ferric chloride, Distilled water & glacial acetic acid. Mix
equal parts of solution A & B.
3. Biebrich Scarlet-Acid Fuchsin Solution: 1% Biebrich Scarlet, 1% Acid
Fuchsin & Glacial acetic acid.
4. Phosphotungstic/Phosphomolybdic Acid solution: Phosphomolybdic acid,
Phosphotungstic acid & distilled water.
5. Aniline Blue solution: Aniline blue dye, Glacial acetic acid & distilled water.
6. 1% Acetic acid solution: Glacial acetic acid & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Rinse slides well in water.
3. Place sections in Bouin’s solution for 1 hour at 56 degrees Celsius. This step is
where we the tissue is be mordanted.
4. Rinse slides in running tap water. This step is necessary for the total removal of the
yellow coloring on the sections.
5. Stain sections in Weigert’s hematoxylin for 10 minutes. This step is where we are
staining the entire nucleus.
6. Rinse slides in running water for 10 minutes. This step helps with the removal of
excess Weigert’s Hematoxylin Stain.
7. Stain slides in Biebrich Scarlet-acid fuchsin for 2 minutes. This step will stain the
cytoplasm of the tissue and other tissue elements.
8. Rinse slides in running tap water to remove any excess staining.
9. Place slides in the Phosphotungstic/Phosphomolybdic acid solution for 10 to 15
minutes. This step removes any staining that has occurred in areas not cytoplasmic.
10. Stain slides in Aniline blue solution for 5 minutes. This is recommended for small
amount of collagen. For large amount of collagen Light green counterstain is
recommended. This step is the collagen staining step.
11. Place slides in 1% acetic acid solution for 3 to 5 minutes. . This is a differentiating
step.
12. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip with
a resinous mounting media.
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 Biebrich-

. Aniline Blue
Weigert’s Scarlet-acid
Hematoxylin fuchsin

Phosphotungstic/P
hosphomolybdic

Dehydration
acid
Water Water
Bouin’s Rinses Rinses

VIII. Results:
Nuclei…..……………………………………………..…….. Black
Cytoplasm, keratin, muscle fibers…………………………... Red
Collagen and Mucous………………………..……………….Blue

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Gomori One-Step Trichrome Stain

I. Purpose of the stain: To identify an increase in collageneous connective tissue fibers

or to differentiate between collagen and smooth muscle fibers.

II. Principle of the stain: In this procedure a plasma stain (Chromophore 2R) and a
connective tissue fiber stain (fast-green FCF, light green, or aniline blue) are combined
in a solution of phosphotungstic acid to which glacial acetic acid has been added.

III. Fixatives: Any well fixed tissue may be used. Bouin solution is used as a mordant to
intensify the color reactions.

IV. Sectioning: Paraffin sections at 4-5 microns.

V. Controls: Every tissue has an internal control, but uterus, small intestine, appendix or
fallopian tube will be good material.

VI. Reagents:
1. Bouin’s Solution: Picric acid saturated, formaldehyde & Glacial acetic acid.
2. Weigert’s Iron Hematoxylin: Solution A: Hematoxylin & 95% Alcohol; &
Solution B: 29% Ferric chloride, Distilled water & glacial acetic acid. Mix equal
parts of solution A & B.
3. Gomori Trichrome stain: Chromophore 2r, Fast Green FCF, light green, or aniline
blue, phosphotungstic acid glacial acetic acid distilled water.
4. 0.5% Acetic acid solution: Glacial acetic acid & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Rinse slides well in water.
3. Place sections in Bouin’s solution for 1 hour at 56 degrees Celsius. This step is
where the tissue is be mordanted.
4. Rinse slides in running tap water. This step is necessary for the total removal of
the yellow coloring on the sections.
5. Stain sections in Weigert’s hematoxylin for 10 minutes. This step is where we are
staining the entire nucleus.
6. Stain sections in Gomori Trichrome solution for 15 to 20 minutes.
7. Place slides in 1% acetic acid solution for 3 to 5 minutes. This is a differentiating
step.
8. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

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 Weigert’s Gomori
Hematoxylin One-Step
Trichrome

Acetic acid
Water Water
Bouin’s Rinses Rinses

VIII. Results:
Nuclei…..……………………………………………………….. Black
Cytoplasm, keratin, muscle fibers………………………………. Red
Collagen and Mucous……………………………………………Green or Blue

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Van Gieson Picric Acid-Acid Fuchsin

I. Purpose of the stain: Considered a primary connective tissue stain, it is used for other
counterstain such as the Verhoeff’s elastic technique, referred to in many laboratories
as the Verhoeff-van Gieson stain.

II. Principle of the stain: This stain will stain collagen selectively by acid fuchsin, an acid
aniline dye. Picric acid provides the acidic pH and acts as a stain for muscle and
cytoplasm. The lower pH is important since high pH will not allow the collagen to
stain.

III. Fixatives: Any well fixed tissue may be used.

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

V. Controls: Every tissue has an internal control, but uterus, small intestine, appendix or
fallopian tube will be good material.

VI. Reagents:
1. Weigert’s Iron Hematoxylin:
Solution A: Hematoxylin & 95% Alcohol
Solution B: Hydrochloric Acid, concentrate, 29% Ferric Chloride solution &
Distilled water.
2. Acid fuchsin, 1%: Acid fuchsin & distilled water
3. Picric Acid saturated: Picric acid & distilled water. It is important that the picric
acid is saturated, if not the collagen will stain of a pale pink to pale orange, and
cytoplasm and muscle may all stain the same color.
4. Van Gieson solution: acid fuchsin, 1% & saturated Picric 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 10 to 20 minutes. Sections should be overstained, as they will
slightly decolorized by the picric acid. 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 van Gieson solution for 5 minutes. This step is where the
collagen will be staining.
5. Place slides in 95% alcohol. This step is to remove the excess non specific
staining.
6. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

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VIII. Results:
Nuclei…………………………………….Black
Collagen.....................................................Brilliant Red
Muscle & cytoplasm..................................Yellow

Van Giesons Stain

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Elastic

Elastic fibers are bundles of proteins (elastin) found in connective tissue and produced by
fibroblasts and smooth muscle cells in arteries. These fibers can stretch up to 1.5 times their
length, and snap back to their original length when relaxed.

Bundles are formed from covalent cross-linking between two amino acids: desmosine and
isodesmosine.

Verhoeff’s Elastic Stain

I. Purpose of the stain: Demonstration of pathologic changes in elastic fibers.

II. Principle of the stain: The tissue is over stain with a solution of hematoxylin-ferric
chloride - iodine solution. Ferric chloride and iodine serve as mordant, but they also
have an oxidizing function in helping the conversion of hematoxylin to hematein.

III. Fixatives: Any well-fixed tissue may be used, 10% Neutral buffered formalin or
Zenker’s is preferred.

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

V. Controls: Aorta embedded on edge or a cross section of a large artery.

VI. Reagents:
1. Lugol’s iodine: Iodine, potassium iodide, distilled water.
2. 10% Ferric chloride: Ferric chloride & Distilled water
3. Alcoholic Hematoxylin, 5%: Hematoxylin & 95% Ethanol.
4. Verhoeff’s Elastic stain: 5% alcoholic hematoxylin, 10% Ferric chloride &
Lugol’s Iodine.
5. Van Gieson Solution: acid fuchsin, 1% & saturated Picric acid.
6. 5% Sodium thiosulfate: Sodium thiosulfate & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place sections in Verhoeff’s Elastic stain for 1 hour. This solution must be
prepared fresh and in the order given, otherwise poor staining may result.
3. Wash slides in 2 changes of distilled water. This step will remove excess
Verhoeff’s Elastic stain.
4. Differentiate section in 2% Ferric chloride until elastic fibers are distinctive and
the background is colorless or light gray. If sections are differentiated too far, re-
stain in the Verhoeff’s elastic stain. This step is a critical step, since we are dealing
with the differentiation of elastic fibers.
5. Rinse slides in distilled water.
6. Place sections in Sodium Thiosulfate for 1 minute. This step will remove any non-
specific staining from the tissue section.
7. Wash slides in running tap water for 5 minutes.

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 8. Counterstain slides in van Gieson solution for 1 minute. This step will allow for
the staining of collagen, muscle and cytoplasm.
9. Differentiate slides in 95% alcohol.
10. Dehydrate slide 100% alcohol, clear in xylene and coverslip with a resinous
mounting media.

5% Sodium
Thiosulfate Van Gieson

Water Water Water

Verhoeff’s Rinses 2% Ferric Rinses Rinses
Elastic Stain chloride
Verhoeff’s Van Gieson on Aorta

VIII. Results:
Elastic fibers......................................................................Blue-black to black
Nuclei................................................................................Blue to black
Collagen............................................................................Red
Other tissue elements........................................................Yellow

Aldehyde Fuchsin Elastic Stain

I. Purpose of the stain: Demonstration of pathologic changes in elastic fibers.

II. Principle of the stain: Hydrochloric acid and paraldehyde are added to an alcoholic
solution of basic fuchsin to form aldehyde fuchsin. Schiff bases are formed by the
aldehyde and the fuchsin, but the affinity of elastic fibers for this solution is not
understood. A number of other tissue elements will also stain with aldehyde fuchsin.
These elements include pancreatic beta cell granules and sulfated mucosubstances.
Staining is intensified by prior oxidation.

III. Fixatives: 10% Neutral buffered formalin, chromate fixatives should be avoided.
Formalin- and Bouin fixed tissues will show a colorless background, and mercury-fixed
tissues show a pale lilac background.

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

V. Controls: Aorta embedded on edge, a cross section of a large artery. Skin also is a
good control.

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 VI. Reagents:
1. Aldehyde fuchsin solution: Pararosaniline, 70% ethyl alcohol, Hydrochloric
acid, Fresh Paraldehyde. Mix well and let it stand for 2 to 3 days or until the
stain is deep purple.
2. Stock Light Green: Light Green Yellowish SF, distilled water & Glacial
Acetic acid.
3. Light Green Working solution: Light green stock & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in aldehyde fuchsin solution for 10 to 40 minutes.
3. Place slides in 70% Alcohol. This step will allow the removal of excess stain.
4. Wash the slides in water and check microscopically for the elastic fibers. If a
deeper stain is desired rinse sections briefly in 70% alcohol and return to the
aldehyde fuchsin.
5. Rinse slides in distilled water
6. Stain slides in working Light green solution for 1 to 2 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.

Water
Rinses

70% Light
Aldyhyde Alcohol Green
fuchsin

VIII. Results:
Elastic fibers..…………………….……...........Deep blue to Purple
Other tissue elements.....……………………... Green

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Movat’s Pentachrome Stain
I. Purpose of the stain: Demonstration of mucin, fibrin, elastic fibers, muscle, and
collagen.

II. Principle of the stain: This stain involves the use of five different stains. Acidic
mucosubstances are stained by Alcian Blue, Iron hematoxylin will stain the elastic
fibers, Crocein scarlet and acid fuchsin will stain the muscle, cell cytoplasm, collagen
and ground substances.

III. Fixatives: 10% Neutral buffered formalin.

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

V. Controls: Lung, skin or colon.

VI. Reagents:
1. 1% Alcian blue: Alcian blue 8X, distilled water & glacial acetic acid.
2. Alkaline alcohol: Ammonium hydroxide, 85% alcohol
3. Iodine-Iodide: Iodine, Potassium Iodide & distilled water.
4. 10% alcoholic hematoxylin: hematoxylin & Absolute alcohol.
5. 10% Ferric chloride: ferric chloride & distilled water.
6. Hematoxylin solution: 10% alcoholic hematoxylin, absolute alcohol, 10%
ferric chloride, & iodine-iodide.
7. 2% Ferric chloride: Ferric chloride & distilled water.
8. 5% Sodium thiosulfate: sodium thiosulfate & distilled water.
9. Crocein scarlet-Acid fuchsin Solution A & Solution B.
10. 5% Phosphotungstic acid solution: phophotungstic acid & distilled water.
11. Alcoholic safran: Safran de Gatinais & absolute alcohol.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain slides in alcian blue for 20 minutes.
3. Wash in running water for 5 minutes.
4. Place slides in alkaline alcohol for 1 hour.
5. Wash slides in running water for 10 minutes.
6. Rinse slides in distilled water.
7. Stain in hematoxylin solution for 15 minutes.
8. Rinse in several changes of distilled water.
9. Differentiate in 2% ferric chloride solution until the elastic fibers contrast
sharply with the background.
10. Rinse slides in distilled water.
11. Place slides in sodium thiosulfate for 1 minute.
12. Wash slides in running tap water for 5 minutes, rinse in distilled water.
13. Stain slides in crocein scarlet-acid fuchsin for 15 minutes.
14. Rinse slides in several changes of distilled water.
15. Rinse slides in 0.5% of acetic acid solution.
16. Place slides in 5% Phosphotungstic acid solution, two changes of 5 minutes
each.
17. Rinse slides in 0.5% of acetic acid solution.

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 18. Rinse slides in three changes of absolute alcohol.
19. Stain slides in alcoholic safran solution for 15 minutes.
20. Rinse slides in three changes of absolute alcohol.
21. Clear slides in two to three changes of xylene and coverslip.

VIII. Results:
Nuclei and elastic fibers............................…..…...Black
Collagen.................................................................Yellow
Ground substances and mucin...............................Blue
Fibrinoid, fibrin......................................................Intense red
Muscle....................................................................Red

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SIVER TECHNIQUES FOR RETICULAR FIBERS
1. Oxidation: Changes of the glycol group to the aldehyde groups.
♦ Phosphomolybdic acid
♦ Potassium permanganate
♦ Periodic acid

2. Sensitization: Usually is a metallic impregnation step.. Impregnation is

the deposition of metallic salts on or around, but not in, the tissue
element to be demonstrated. The exact chemical reaction is not
understood. The sensitizing metal is then replaced by silver.
♦ Uranyl nitrate
♦ ferric ammonium sulfate
♦ dilute solution of silver nitrate

3. Silver Impregnation: Involves treating tissue with an ammoniacal or

diamine silver complex.

4. Reduction: The silver is reduced into a visible metallic state.

♦ Formaldehyde
♦
5. Toning: Term used when bound metallic silver is treated and the color
of the impregnated component changes from brown to black. The
metallic silver is being replaced by metallic gold.
♦ Gold Chloride
♦
6. Unreduced silver: This step prevents any non specific bound silver
remaining in the section from being reduced by a later exposure to
light.
♦ Sodium Thiosulfate (Hypo)

7. Counterstain: May or may not be used, is up to the laboratory

procedures.
♦ Nuclear Fast Red (If counterstain is used)

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Gomori Stain for Reticular Fibers
I. Purpose of the stain: Demonstration of reticular fibers in tissue sections. A change of
normal pattern as seen in some liver diseases, is also an important diagnostic finding.

II. Principle of the stain: The tissue will be oxidize converting the sugar group to
aldehyde followed by a sensitizing step for metallic salts. Impregnation of with silver
will follow, then it will be reduced to a metallic visible state by formaldehyde. Gold
chloride is applied as a toner which will change the bound silver from a brown color to
a black color. A hypo solution will follow in order to remove any unreduced silver, and
if desired a counterstain is applied.

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

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

V. Controls: Liver is a very good control tissue.

VI. Reagents:
1. 10% Silver nitrate: silver nitrate & distilled water.
2. Potassium hydroxide solution: potassium hydroxide & distilled water.
3. Ammoniacal silver solution: 10% silver nitrate & potassium hydroxide.
Concentrate ammonium hydroxide is added drop by drop until it becomes
slightly cloudy and then clear again.
4. 0.5% Potassium permanganate: potassium permanganate & distilled water.
5. 2% potassium metabisulfite: Potassium metabisulfite & distilled water.
6. 2% ferric ammonium sulfate: Ferric ammonium sulfate & distilled water.
7. Formalin solution: Formaldehyde, 37 to 40% & distilled water.
8. 0.2% Gold chloride: Gold chloride and distilled water.
9. 2% Sodium thiosulfate: Sodium thiosulfate & distilled water.
10. Nuclear Fast Red

VI. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in 0.5% potassium permanganate solution for 1 minute. This step is
changing the glycol group to aldehyde group.
3. Rinse slides in tap water for 2 minutes.
4. Place slides in 2% potassium metabisulfite for 1 minute. This step will remove
the purple color from the tissue section.
5. Rinse slides in tap water for 2 minutes.
6. Place section in 2% ferric ammonium sulfate for 1 minute. This is the
sensitization step.
7. Rinse slides in tap water for 2 minutes. Followed by two changes of distilled
water.
8. Place section in the Ammoniacal silver for 1 minute. This is the impregnation
step.
9. Rinse slides in distilled water for 20 seconds.
10. Place slides in formalin solution for 3 minutes. This is the reduction step.
11. Rinse slides in tap water for 3 minutes.

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 12. Place slides in 0.2% gold chloride solution for 10 minutes. This is the toning
step.
13. Rinse slides in distilled water.
14. Place slides in potassium metabisulfite for 1 minute.
15. Place slides in 2% sodium thiosulfate. This is the step where the unreduced
silver is removed.
16. Rinse slides in tap water for 2 minutes.
17. Counterstain if desired with Nuclear Fast Red for 5 minutes.
18. Rinse well in tap water.
19. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

Sodium Thiosulfate
Formaldehyde
Silver Nitrate

Gold Chloride

Nuclear fast red

Potassium permanganate

Potassium metabisulfite

Ferric Ammonium Sulfate

Oxidation Sensitization Impregnation Reducer Toning Hypo Counterstain

VII. Results:
Reticulin fibers.........................................................Black
Collagen....................................................................Taupe

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Gordon & Sweets Stain for Reticular Fibers

I. Purpose of the stain: Demonstration of reticular fibers in tissue sections. A change of

normal pattern as seen in some liver diseases, is also an important diagnostic finding.

II. Principle of the stain: The tissue will be oxidize converting the sugar group to
aldehyde followed by a sensitizing step for metallic salts. Impregnation of with silver
will follow, then it will be reduced to a metallic visible state by formaldehyde. Gold
chloride is applied as a toner which will change the bound silver from a brown color to
a black color. A hypo solution will follow in order to remove any unreduced silver, and
if desired a counterstain is applied.

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

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

V. Controls: Liver is a very good control tissue.

VI. Reagents:
1. 10% Silver nitrate: silver nitrate & distilled water.
2. 3% Sodium hydroxide solution: sodium hydroxide & distilled water.
3. Ammoniacal silver solution: 10% silver nitrate & 3% sodium hydroxide.
Concentrate ammonium hydroxide is added drop by drop until it becomes
slightly cloudy and then clear again.
4. 1% Potassium permanganate: potassium permanganate & distilled water.
5. 2.5% ferric ammonium sulfate: Ferric ammonium sulfate & distilled water.
6. Formalin solution: Formaldehyde, 37 to 40% & distilled water.
7. 0.2% Gold chloride: Gold chloride and distilled water.
8. 2% Sodium thiosulfate: Sodium thiosulfate & distilled water.
9. Nuclear Fast Red

VIII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in 0.5% potassium permanganate solution for 1 minute. This step is
changing the glycol group to aldehyde group.
3. Rinse slides in tap water for 2 minutes.
4. Place slides in 1% oxalic acid for 2 minutes or until sections are colorless. This
step will remove the purple color from the tissue section.
5. Rinse slides in tap water for 2 minutes.
6. Place section in 2.5% ferric ammonium sulfate for 15 minute. This is the
sensitization step.
7. Rinse slides in tap water for 2 minutes. Followed by two changes of distilled
water.
8. Place section in the Ammoniacal silver for 2 minute. This is the impregnation
step.
9. Rinse slides in distilled water for 20 seconds.
10. Place slides in formalin solution for 2 minutes. This is the reduction step.
11. Rinse slides in tap water for 3 minutes.
12. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.
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 13. Place slides in 2% sodium thiosulfate for 1 minute. This is the step where the
unreduced silver is removed.
14. Rinse slides in distilled water.
15. Place slides in 0.2% gold chloride solution for 2 minutes. This is the toning step.
16. Counterstain if desired with Nuclear Fast Red for 5 minutes.
17. Rinse well in tap water.
18. Dehydrate slide with 95% alcohol, 100% alcohol, clear in xylene and coverslip
with a resinous mounting media.

Sodium Thiosulfate
Formaldehyde
Silver Nitrate

Gold Chloride

Nuclear fast red

Oxalic Acid
Potassium permanganate

Ferric Ammonium Sulfate

Oxidation Bleaching Sensitization Impregnation Reducer Hypo Toning Counterstain

IX. Results:
Reticulin fibers.........................................................Black
Background………………………………………..Red

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 PTAH for Cross-Striations and Fibrin
I. Purpose of the stain: For the demonstration of cross-striations and fibrin. Cross-striations
are a diagnosis feature of rhabdomyosarcomas or tumors arising from striated muscles.

II. Principle of the stain: This stain has been referred to as a polychrome stain because one
solution can give two major colors.

III. Fixatives: Zenker’s solution is preferred, but 10% NBF can be used.

IV. Sectioning: Cut paraffin sections from 4 to 6 microns.

V. Controls: Skeletal or cardiac muscle for cross-striations or a section with fibrin for fibrin
demonstration, or cerebral cortex for glial fibers demonstration.

VI. Reagents:
1. PTAH solution: Hematoxylin, Phosphotungstic acid, distilled water.
2. Gram Iodine: Iodine, Potassium Iodide & distilled water.
3. 5% Sodium thiosulfate & distilled water.
4. 0.25% Potassium Permanganate: potassium permanganate & distilled water.
5. 5% Oxalic acid: oxalic acid & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. If tissues are formalin fixed, mordant in Zenker’s solution containing 5% acetic
acid overnight at room temp.
3. Place slides in tap water.
4. Place slides in Gram Iodine for 15 minutes.
5. Rinse slides in tap water.
6. Place slides in 5% sodium thiosulfate solution for 3 minutes.
7. Rinse slides in tap water for 10 minutes.
8. Place sections in 0.25% potassium permanganate for 5 minutes.
9. Rinse slides in tap water.
10. Place slides in 5% Oxalic acid for 1 minute.
11. Wash slides in running tap water for 10 minutes.
12. Stain slides in PTAH solution overnight at room temperature.
13. Dehydrate rapidly through two changes of 95% alcohol, 100% alcohol, clear in
xylene and coverslip.

XI. Results:
Cross striations and Fibrin.......................................................Blue
Nuclei......................................................................................Blue
Collagen..................................................................................Red Brown

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Jones Method (Periodic Acid Methenamine Silver)

I. Purpose of the stain: This procedure best delineates the glomerular basement
membrane.

II. Principle of the stain: Methenamine silver demonstrates the carbohydrate

component of basement membrane by oxidizing the carbohydrate to an aldehyde. The
tissue will be oxidize converting the sugar group to aldehyde followed by a
sensitizing step for metallic salts. Impregnation of with silver will follow. Gold
chloride is applied as a toner which will change the bound silver from a brown color
to a black color. A hypo solution will follow in order to remove any unreduced
silver, and light green is applied as a counterstain.

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

IV. Sectioning: Cut paraffin sections from 2 microns.

V. Controls: Kidney has an internal control.

VI. Reagents:
1. Stock Methenamine Silver: 3% methenamine solution & 5% silver solution.
2. 5% Borax solution: Sodium borate & distilled water.
3. Working Silver: Stock methenamine silver, Borax solution and distilled water.
4. 1% Periodic acid solution: Periodic acid & distilled water.
5. 0.02% Gold Chloride Solution: Gold chloride & distilled water.
6. Light Green Solution: Stock light green solution & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in 1% Periodic acid solution for 15 minutes. This is the oxidizing
step where is changing the glycol group to aldehyde group.
3. Rinse slides in tap water for 2 minutes.
4. Place section in the Methenamine silver for 15 to 20 minute. This is the
impregnation step.
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 5. Rinse slides in heated distilled water.
6. Place slides in 0.2% gold chloride solution for 2 minutes. This is the toning step.
7. Rinse slides in distilled water.
8. Place slides in 2% sodium thiosulfate for 1 minute. This is the step where the
unreduced silver is removed.
9. Rinse slides in tap water.
10. Counterstain in light green solution for 1 1/2 minutes. Dehydrate slide with 95%
alcohol, 100% alcohol, clear in xylene and coverslip with a resinous mounting
media.

Pe Sil Go Li
So
rio ve ld gh
di
di r Ch u t
c Ni lor Gr
m
aci tra ide ee
Th
d te ios n
ulf
ate

Oxidation Impregnation Toning Hypo Counterstain

VIII. Results:
Basement Membranes.........................................................Black
Background.........................................................................Green

23
 TECHNIQUE FOR LIPIDS

Oil Red O Method for Neutral Fats

I. Purpose of the stain: Demonstration of neutral lipids in frozen sections.

II. Principle of the stain: This is a physical stain and the dye used must be more soluble
in the tissue lipid than in the solvent in which it is dissolved. Must not be water soluble.
Must be strongly colored, and must act with tissue constituents only by solution.

III. Fixatives: 10% NBF or calcium formol, because of lipid dissolving abilities, no
alcoholic fixatives should be used.

IV. Sectioning: Cut frozen sections at 10 microns.

V. Controls: Most tissue contains fats, normally no control is needed.

VI. Reagents:
1. Oil Red O Stock solution: Oil Red O, Isopropanol, 98%.
2. Oil red O Working solution: Oil Red O Stock solution & Distilled water. Mix
well and let stand for 10 minutes before using.

VII. Procedure:
1. Cut frozen sections and fix in 40% formaldehyde for 1 minute, and wash well in
tap water. Blot off excess water. If the tissue has been previously fixed, then
there is no need of fixation.
2. Stain slides in Working Oil Red O solution for 10 minutes.
3. Wash sections in tap water.
4. Stain slides in Harris’ hematoxylin with acetic acid for 1 minute.. This
step will stain all the nuclear details of the tissue.
5. Wash slides in running tap water for several minutes.
6. Blue slides in ammonium water.
7. Wash slides in tap water.
8. Mount sections with an aqueous mounting medium.
9. Seal edges with fingernail polish.

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 Water Rinses
Formaldehyde Oil Red O Harris
post fixing Hematoxylin
/Bluing

IX. Results:
Fat .........................................................................Intense Red
Nuclei.....................................................................Blue

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Osmium Tetroxide Paraffin Procedure for Fat
I. Purpose of the stain: Demonstration of fat by a method that allows paraffin
embedded of the tissue.

II. Principle of the stain: Osmium tetroxide chemically binds with the fat blackening it
in the process. This is the only method for demonstration of fat on paraffin sections.

III. Fixatives: 10% NBF.

IV. Sectioning: Stain the wet tissue with the Osmium tetroxide, then process the tissue.
The section must be no thicker than 2 mm or the osmium will not penetrate.

V. Controls: Most tissue contains fats, normally no control is needed.

VI. Reagents:
1. 1% Osmium tetroxide solution: osmium tetroxide & distilled water.
2. 0.5% Periodic acid solution: periodic acid & distilled water.

VII. Procedure:
1. Trim formalin fixed tissue to 2 mm thickness, then wash thoroughly for at least
30 minutes.
2. Rinse the tissue well in distilled water.
3. Place the section in a small quantity of Osmium Tetroxide solution and leave for
at least 1 to 2 hours.
4. Rinse slides in two changes of distilled water for 15 minutes each.
5. Differentiate slides by placing them in the 0.5% Periodic acid solution for 30
minutes.
6. Wash slides in tap water for 30 minutes.
7. Process routinely, beginning with 70% alcohol, embed as usual.
8. Cut paraffin sections 4-5 microns, pick on slides and dry as normal.
9. Deparaffinize and hydrate as usual.
10. Counterstain with hematoxylin, Masson Trichrome.
11. Dehydrate, clear and mount slides as normal.

IX. Results:
Fat .............................................................. Black
Other tissue elements ................................. According to method used

26
Toluidine Blue for Mast cells
I. Purpose of the stain: Demonstration of mast cells in tissue.

II. Principle of the stain: Mast cells will stain metachromatically (A different color
from the dye) with Toluidine blue.

III. Fixatives: 10% NBF.

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

V. Controls: Sections containing mast cells.

VI. Reagents:
1. Toluidine Blue Solution: Toluidine blue & distilled water.

VII. Procedure:
1. Deparaffinize and hydrate slides to water.
2. Stain slides in Toluidine blue solution for 10 minutes.
3. Rinse sections in distilled water.
4. Quickly dehydrate in through 95% and absolute alcohol.
5. Clear in xylene and mount with synthetic resin.

VIII. Results:
Mast cells............................................... Deep violet
Background............................................ Blue

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