HISTOLOGY

LECTURE # 30

INTRODUCTION TO SPECIAL STAINS TECHNIQUES:

PIGMENTS & MINERALS STAINING

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 pigment & mineral staining.

b) Learn the various methods of stain demonstration.

c) Learn the classification for iron, urate crystals, copper and other granule substances staining.

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

PIGMENT & MINERALS

Types of Pigments

• Endogenous Pigment
• Exogenous Pigment
• Hematogeneous pigment
• Anthracotic pigment
• endogenous, non-hematogeneous pigment
• Mineral
 Definitions:
Pigment - coloring agents deposited, frequently as cytoplasmic inclusions or granules in cells and tissue.
Three majors groups:
• Artifact Pigments
• Exogenous pigments
• Endogenous Hematogeneous pigments

I. Artifact Pigments
These pigments are deposited in the tissue as a result of a chemical action during processing, and
most commonly result from fixation. They normally lie on top of the tissue and not within the cell.
Examples:
1. Mercury - any mercury containing fixative
2. Formalin - acidic formaldehyde solution
3. Chrome pigments - potassium dichromate solutions

II. Removal of Pigments

1. Mercury Pigments - Iodine solution followed by a Sodium Thiosulfate solution
2. Chrome Pigments - Washed thoroughly in water before dehydration
3. Formalin Pigments - alcoholic picric acid or alkalis, resistant to strong acids

III. Exogenous Pigments

– These pigments are formed externally and then taken into the body by various routes.
A. Carbon (Anthracotic pigment) - black pigment seen in lung and lymph nodes. It
resists bleach and extraction procedures. These are demonstrated with silver stains.

B. Asbestos fibers - birefringement fibers of magnesium silicate that may be found in

people exposed to large quantities of asbestos. This can lead to mesothelioma.
After inhalation the fibers become coated with an iron-containing protein and are
known as asbestos bodies. These are demonstrated with the Prussian Blue reaction
for iron.

C. Tattoo pigments - found in tattooed skin, but occasionally found in associated

lymph nodes. They include a series of organic and inorganic pigments.

IV. Endogenous Hematogeneous Pigments

¾ These pigments are formed within the blood in the body.
¾ They are classified as Hematogeneous (from blood) or non-Hematogeneous.
A. HEMATOGENEOUS
1. Hemoglobin - is a conjugated protein that is found normally in red blood
cells and that is responsible for transporting oxygen from the lungs to other
parts of the body.
a) Staining:
1) Eosin staining
2) Okajima Stain

2. Hemosiderin - large amount are found only in pathologic conditions. If the

production and destruction of RBC are not balanced, there may be increased
deposition of hemosiderin in tissue
• Hemochromatosis - disease caused by excessive absorption of
dietary iron, is also characterized by excessive hemosiderin deposits.
a) Staining:
1) Prussian Blue Reaction
 3. Bile pigments - Biliverdin also results from destruction of RBC’s and
further breakdown of the heme portion of hemoglobin.

a) Biliverdin - is transported to the liver, where it undergoes

reduction to bilirubin, which is not normally deposited in tissue
but is removed from circulation by the liver and the secreted as
one of the components of bile.
b) Jaundice - caused by the obstruction of normal bile flow.
c) Staining:
1) Hall’s Stain for Bile

4. Hematoidin - similar to bilirubin and oxidized to biliverdin by bile

demonstrating techniques. It’s formed in tissue as a result of hemorrhage
and reduced oxygen tension.

Endogeneous Pigments

Hematogeneous Non-Hematogeneous

Hemoglobin Lipidic Non- Lipidic

Lipofuchsin Ceroid Melanin

Globin Heme

(protein part) (pigment part)

Amino acid pool

Bile pigments
Iron
Biliverdin
Reused in new Deposited in tissue
hemoglobin as hemosiderin
Bilirubin further breakdown products
synthesis
V. Endogenous Non-hematogeneous Pigments
¾ These pigments are not from blood.
¾ NON-HEMATOGENEOUS
Non lipidic pigments
1. Melanin - mixture of substances bound to proteins. Brown to black pigment
present normally in the hair, skin, retina, iris, and certain parts of the central
nervous system.

Synthesis of Melanin

Oxidation by

tyrosine (amino acid) tyrosine (enzyme) DOPA (3,4-dihydroxy phenylalanine)

dopa further oxidation melanin

through several steps

2. This reaction allows the histochemical demonstration of melanocytes, the cells

responsible for the formation of melanin pigments.

a) Melanin bleaching = when melanin is present in large amounts, cell details

may be obscured. An oxidizing agent is used in order to remove the excess
melanin.
» 10% Potassium permanganate followed by Oxalic acid.

lipidic pigments
1. Lipofuchsin - referred to as “wear and tear” pigment, collects in the more
permanent cells: heart, liver, neurons. It is a yellow-brown pigment that stains in
Oil red O, Sudan Black B, and PAS reaction.
2. Ceroid - is a brownish-yellow pigment described in hepatocytes and macrophages
of rats with experimentally induced cirrhosis of the liver. Rarely seen in humans.
1) Stains:
a) Oil Red O
b) Sudan Black B
 VI. Endogenous Deposits
A. Urates, are deposited in tissue (gouty tophi) or around joints in person suffering from gout.
B. Gout - a disorder in uric acid metabolism
1. Sodium urate are very soluble in water
2. dissolves if fixed in routine aqueous fixatives
3. alcoholic fixatives are recommended
4. Birefringent and identified by polarizing microscope
a) Stain: Argentaffin reaction - Methenamine silver

VII. Minerals
¾ Metallic and non metallic ions necessary for growth and other bodily functions.
A. Ions demonstrated with special stains are:
• Calcium
• Ferrous/Ferric
• Cupric
• Phosphate
• Carbonate
B. Metallic elements are pathologically in tissue:
ƒ Silver
ƒ Lead
ƒ Copper
ƒ Gold
C. Microincineration - method employed to study inorganic substances found in tissue.
Process: Slides are cut at 4 µm and mounted on high temperature glass, the slides are
incinerated up to 650oC, once completed slides are allowed to cool, then removed from the
oven, coverslipped with glycerol mounting medium and microscopically examined.

VIII. Types of Cytoplasmic Granules

• Adrenal chromaffin cells
• Pancreatic endocrine cells
• gastrointestinal enterochromaffin cells
• “C” cells of the thyroid
• Pituitary cells
• Neuroendocrine cells

Demonstration:
¾ Argyrophilic - have the ability to be impregnated by silver, but do not have the ability to
reduce the silver to a metallic form without the help of a reducing agent.
(NEED A REDUCER)

¾ Argentaffin - They have the ability to be impregnated by silver and the ability to reduce the
silver to a metallic form without the need of a reducing agent.
(NO REDUCER NEEDED)

***Argentaffin cells will give a positive reaction with argyrophil techniques, but argyrophil cells
will not yield positive results with argentaffin techniques ***
 Special Staining Techniques
Prussian Blue Stain for Ferric Ion (Fe+3)
Purpose: The detection of ferric iron in tissue. Found normally in spleen and bone marrow.

Principle: Detects the loosely bound protein complexes (As in hemosiderin). Strong bound iron will not
react. Sections are treated with an acidic solution of potassium ferrocyanide and any ferric ion present will
react in a bright blue pigment called Prussian Blue.

Fixative: Alcohol or 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections.

Control: a section containing ferric iron must be used.

Reagents: 2% Potassium Ferrocyanide: Potassium ferrocyanide & distilled water

2% Hydrochloric acid Solution: Concentrated hydrochloric acid & distilled water
Nuclear Fast Red solution: Nuclear fast Red, Aluminum sulfate, distilled water & Thymol
Procedure:
1. Deparaffinize and hydrate slides to water
2. Place slides in a freshly prepared solution of a 50:50 mixture of 2% potassium ferrocyanide & 2%
hydrochloric acid for 20 minutes at 60oC.
3. Wash slides thoroughly in water.
4. Counterstain slides in Nuclear fast Red solution for 5 minutes.
5. Rinse slides in running tap water for 1 minute.
6. Dehydrate sections in 95% alcohol and two changes of absolute alcohol.
7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Nuclei & hemofuchsin …………………………………………………...…Bright Red
Hemosiderin (iron)……………………………………………………….….Blue
Background…………………………………………………………….…….Pink
Turnbull’s Blue Stain for Ferrous Iron (Fe+2)
Purpose: The detection of ferrous iron in tissue.

Principle: Detects the ferrous iron in tissue. Sections are treated with an acidic solution of potassium
ferricyanide and any ferrous iron present will react to form an insoluble bright blue pigment called
Turnbull’s blue (ferrous ferricyanide).

Fixative: Alcohol or 10% Neutral buffered formalin

Technique: 4 to 5 µm paraffin sections

Control: a section containing ferrous iron must be used.

Reagents: 0.06N Hydrochloric acid: Hydrochloric acid & Distilled water

Potassium Ferricyanide Staining Solution:: Potassium ferricyanide, 0.06N Hydrochloric acid
Prepare fresh before use.
1% Acetic acid: glacial Acetic acid & distilled water
Nuclear Fast Red solution: Nuclear fast Red, Aluminum sulfate, distilled water & Thymol
Procedure:
1. Deparaffinize and hydrate slides to water
2. Place slides in a freshly prepared ferricyanide staining solution and stain for 1 hour at Room
temperature.
3. Wash slides in 1% acetic acid thoroughly in water.
4. Counterstain slides in Nuclear fast Red solution for 5 minutes.
5. Rinse slides in running tap water for 1 minute.
6. Dehydrate sections in 95% alcohol and two changes of absolute alcohol.
7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Ferrous iron …………………………..……...………………………………………………Bright Red
Background…………………………..……………………………………………………….Pink

ASBESTOS FIBERS ASBESTOS FIBERS

Schmorl’s Technique for Reducing Substances
Purpose: The detection of ferrous iron in tissue.

Principle: Detects the ferrous iron in tissue. Sections are treated with an acidic solution of potassium
ferricyanide and any ferrous iron present will react to form an insoluble bright blue pigment called
Turnbull’s blue (ferrous ferricyanide).

Fixative: 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections.

Control: a section containing melanin or argentaffin granules.

Reagents:
1% Stock ferric chloride: Ferric chloride & distilled water
0.1% Potassium Ferricyanide: Potassium ferricyanide & distilled water
Ferric chloride-Potassium ferrocyanide: 1% ferric chloride & 0.1% Potassium ferricyanide, pH to
2.4 with 1N hydrochloric acid
Mayer’s Mucicarmine Solution
Metanil Yellow: Metanil yellow, Distilled water & glacial acetic acid

Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in a freshly prepared ferric chloride - potassium ferricyanide solution and stain for 3
changes of 7 minutes each.
3. Rinse sections in distilled water.
4. Stain slides in working Mayer’s Mucicarmine for 1 hour.
5. Rinse slides rapidly in distilled water.
6. Counterstain slides in Metanil yellow for a few seconds.
7. Rinse slides rapidly in distilled water.
8. Dehydrate sections in 95% alcohol and two changes of absolute alcohol.
9. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Reducing Substances.………………………………………..………………Blue-green
Goblet Cells, mucin…………………………………………………….……Rose
Background………………………………………………………………….Yellow-green
Fontana Masson for Melanin and Argentaffin granules
Purpose: Demonstration of argentaffin substances such as melanin, argentaffin granules of carcinoid
tumors, and some neurosecretory granules.

Principle: Some processes are argentaffin meaning they have the ability to bind silver from a silver
solution and to reduce it to the visible metallic silver without the need for a separate reducing agent.

Fixative: 10% Neutral buffered formalin, alcoholic fixative should be avoided since it tends to dissolves
the argentaffin granules.

Technique: 4 to 5 µm paraffin sections.

Control: a section of skin for melanin, appendix or small intestine for argentaffin granules.

Reagents:
10% Silver Nitrate
Fontana Silver Solution: 10% Silver Nitrate & Ammonium hydroxide
Gold Chloride
Sodium Thiosulfate
Nuclear Fast Red

Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in silver nitrate solution in water bath at 56 degrees. For 1 hour.
3. Rinse sections in distilled water.
4. Immerse slides in gold chloride for 10 minutes.
5. Rinse slides rapidly in distilled water.
6. Place slides in Sodium thiosulfate for 5 minutes.
7. Rinse slides in distilled water.
8. Counterstain slides in Nuclear fast Red for 5 minutes.
9. Wash slides for at least 1 minute in distilled water.
10. Dehydrate sections in 95% alcohol and two changes of absolute alcohol
11. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Melanin .………………………….………………………….............................…Black
Argentaffin granules………………………………………………………………Black
Nuclei……………………………….................………………………………….Pink
Grimelius’ Argyrophil Stain
Purpose: Demonstration of argyrophil granules in neurosecretory tumors.

Principle: Some processes are argyrophilic meaning they need a reducing agent in order to bring the
silver to a visible metallic form.

Fixative: 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections

Control: an argyrophilic-positive carcinoid tumor is preferred, but a section of small intestine can be
used. Glassware should be chemically cleaned.

Reagents:
1% Silver Nitrate: Silver nitrate & Distilled water
0.2M Acetic acid: Glacial Acetic acid & Distilled water
0.2M Sodium Acetate: Sodium acetate & Distilled water
Working Silver Solution: Acetic acid-acetate Buffer, 1% Silver nitrate & Distilled water
Reducing Solution: Hydroquinone, Sodium sulfite & Distilled water
Nuclear Fast Red: Nuclear Fast Red, Distilled water & Thymol

Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in working silver nitrate solution in water bath at 60 degrees for 1 hour.
3. Drain slides briefly.
4. Place slides in freshly prepared reducing solution preheated to 40 - 50 degrees Celsius for 1 minute.
5. Rinse slides well in distilled water.
6. Repeat step 2 for 10 minutes.
7. Drain slides briefly and place in reducing solution again for 1 minute.
8. Rinse slides in distilled water for 2 minutes.
9. Counterstain slides in Nuclear Fast Red solution for 5 minutes.
10. Dehydrate sections in 95% alcohol and two changes of absolute alcohol
11. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Argyrophil granules.……………………..........................................................…Dark brown to black
Argentaffin granules…………………………………………………………..…Dark brown to black
Nuclei……………………………...............…………………………………… Red
Background……………………………………………………...………………Pale yellow-brown
Churukian-Schenk Method for Argyrophil Granules
Purpose: Demonstration of argyrophil granules in neurosecretory tumors.

Principle: Some processes are argyrophilic meaning they need a reducing agent in order to bring the
silver to a visible metallic form.

Fixative: 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections

Control: an argyrophilic-positive carcinoid tumor is preferred, but a section of small intestine can be
used. Glassware should be chemically cleaned.

Reagents:
0.3% Citric acid: Citric acid & distilled water
Acidified water: Distilled water and enough 0.3% Citric acid solution.
0.5% Silver Nitrate: Silver nitrate & Distilled water
Reducing Solution: Hydroquinone, Sodium sulfite &
Distilled water (Prepare just before use)
Nuclear Fast Red: Nuclear Fast Red, Distilled water & Thymol

Procedure:
1. Deparaffinize slides through xylene and alcohol's to acidified water, pH 4.0 to 4.2.
2. Place slides in 0.5% Silver nitrate solution in a 43 degrees water bath for 2 minutes. Transfer to a 58
degrees water bath for 2 hours.
3. Rinse slides in three changes of distilled water.
4. Transfer slides to reducing solution already heated in the 58 degrees water bath for 30 to 60 minutes.
5. Rinse sections in three changes of distilled water.
6. Return sections to the same silver nitrate of step 2 at 58 degrees for 10 minutes.
7. Rinse slides in three changes of distilled water.
8. Place slides in the same reducing solution of step 4 at 58 degrees for 5 minutes.
9. Rinse slides in three changes of distilled water.
10. Counterstain slides in Nuclear Fast Red solution for 3 minute.
11. Rinse slides in three changes of distilled water.
12. Dehydrate sections in two changes of 95% alcohol and two changes of absolute alcohol.
13. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Argyrophil granules.……….……………………….……………........................…Black
Argentaffin granules……………………………………………………………..…Black
Nuclei………………………………………...............…………………………… Red
Background………………………………….……………………...………………Yellow-brown
Gomori’s Methenamine Silver Method for Urates
Purpose: Demonstration of urates in tissue.

Principle: Urates are stained black by this method, it is presumed that it reacts with the silver, which is
then reduced to its metallic form.

Fixative: Absolute alcohol is required.

Technique: 4 to 5 µm paraffin sections

Control: a section containing urate crystals.

Reagents:
5% Silver Nitrate: Silver nitrate & Distilled water
3% Methenamine Solution: Methenamine & distilled water
Stock Methenamine-Silver Solution: 5% silver Nitrate & 3% Methenamine solution.
5% Sodium Borate (Borax): Sodium borate & distilled water
3% Sodium thiosulfate: sodium thiosulfate & distilled water
Stock light green solution: Light green, distilled water & glacial acetic acid
Working Light green: Stock light green and distilled water.

Procedure:
1. Deparaffinize slides and rinse in several changes of absolute alcohol. Do not hydrate slides.
2. Place sections in working methenamine-silver solution which has been preheated at 60 degrees.
3. Incubate slides for 30 minutes at 60 degrees. The urate crystal should turn black.
4. Rinse slides in distilled water.
5. Place sections in 3% sodium thiosulfate (hypo) for 5 minutes.
6. Wash slides in running tap water for 2 to3 minutes.
7. Rinse slides in several changes of distilled water.
8. Counterstain slides in working light green solution for 1 1/2 to 2 minutes.
9. Dehydrate sections in two changes of 95% alcohol and two changes of absolute alcohol.
10. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Urates……………………………………………...……………………........................…Black
Background……………………………………...………………………...………………Green
Bile Stain (Hall)
Purpose: Demonstration of bilirubin in tissue.

Principle: Bilirubin is oxidized to biliverdin in an acid medium. This oxidation reaction is rapidly
accomplished by ferric chloride in trichloroacetic acid medium.

Fixative: 10% Neutral buffered formalin

Technique: 4 to 5 µm paraffin sections

Control: a section containing bile must be used as a control.

Reagents:
10% Ferric chloride: Ferric chloride & Distilled water
Fouchet’s Reagent: Trichloroacetic acid, 10% Ferric chloride & distilled water.
Van Gieson’s solution: 1% acid fuchsin & Saturated Picric acid.

Procedure:
1. Deparaffinize and hydrate slides to distilled water.
2. Wash sections well in distilled water.
3. Stain sections in freshly prepared Fouchet’s solution for 5 minutes.
4. Wash slides in tap water, then rinse in distilled water.
5. Stain sections in Van Gieson’s solution for 5 minutes.
6. Place slides directly into 95% alcohol and rinse well.
7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Bile or Bilirubin……………………………………………...…….…Emerald green to olive drab
Background…………………………………………...………………Yellow
Von Kossa Calcium Stain
Purpose: To identify the presence of calcium in tissue..

Principle: This is a chemical reaction, even though is going in an indirect way of detecting calcium. The
silver reacts with the anions, primarily carbonate and phosphate, of the calcium salts. Bright light reduces
the silver salt to metallic silver and unreduced silver is removed with sodium thiosulfate.

Fixative: 10% Neutral buffered formalin. Alcohols are preferred.

Technique: 4 to 5 µm paraffin sections

Control: a section containing calcium must be used as a control.

Reagents:
5% Silver nitrate: Silver nitrate & distilled water
5% Sodium thiosulfate: Sodium thiosulfate & distilled water
Nuclear Fast Red: Nuclear fast Red, distilled water & Thymol

Procedure:
1. Deparaffinize and hydrate slides to distilled water.
2. Place sections in silver nitrate solution and expose to bright sunlight for 10 to 20 minutes. If the
day is overcast, longer incubation will be necessary. Check the slides periodically and stop the
reaction when the calcium salts are brown-black.
3. Wash sections well in distilled water.
4. Place slides in Sodium thiosulfate for 2 to 3 minutes.
5. Wash slides well in distilled water.
6. Counterstain slides in Nuclear-fast red for 5 minutes.
7. Wash sections well in distilled water.
8. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Calcium salts……………………………………………....…….…Black
Background…………………………………………...………………Red
Alizarin Red S Calcium Stain
Purpose: To identify the presence of calcium in tissue..

Principle: Alizarin red S will react with the cations calcium, magnesium, manganese, barium, and
strontium, but normally only calcium is present in tissue in sufficient quantities for demonstration.
Calcium forms an alizarin red s-calcium complex in a chelation process.

Fixative: Alcoholic fixatives or 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections

Control: a section containing calcium must be used as a control.

Reagents:
Alizarin Red S Staining solution:
Alizarin Red S & Distilled water; Mix the solution and pH to 4.1 to 4.3 with 0.5% ammonium
hydroxide. The pH is critical.

Procedure:
1. Deparaffinize and hydrate slides to 50% alcohol.
2. Rinse slides rapidly in distilled water.
3. Place sections in alizarin red S staining solution. Check the reaction microscopically and remove
slides when an orange-red lake forms (30 seconds to 5 minutes).
4. Shake off excess dye and carefully blot the sections.
5. Dehydrate in acetone (10 to 20 seconds) and in acetone-xylene mixture (10 to 20 seconds).
6. Clear slides in three changes of xylene and mount in a synthetic
resin mounting media.

Results:
Calcium deposits……………………………….………………………....…….…Orange-red

Paraffin Section Wet (Cytology) Section

Rhodanine Method for Copper
Purpose: To detect copper in tissue, especially in liver in Wilson’s Disease.

Principle: This procedure is more sensitive than the rubeanic acid method; however, it has suggested that
Rhodanine demonstrates the protein to which the copper binds rather than the copper itself. Therefore,
although considered more sensitive, it may be less specific than the rubeanic acid methods and false-
positive results may be obtained.

Fixative: 10% Neutral buffered formalin.

Technique: 4 to 5 µm paraffin sections

Control: a section containing copper must be used as a control.

Reagents:
Saturated Rhodanine solution: Rhodanine & absolute alcohol
Working Rhodanine solution: Saturated Rhodanine solution & distilled water. Filter before use.
Diluted Mayer’s Hematoxylin: Mayer’s hematoxylin & distilled water.
0.5% Sodium borate: Sodium borate & distilled water.

Procedure:
1. Deparaffinize and hydrate slides to distilled water.
2. Using plastic coplin jars, place slides in 50 mL of working Rhodanine solution. Leave 18 hours at
37 degrees.
3. Rinse slides well in distilled water.
4. Stain slides in Mayer’s hematoxylin for 10 minutes.
5. Rinse slides in distilled water.
6. Rinse slides quickly in 0.5% sodium borate solution.
7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media.

Results:
Copper………………………….………………………………....…….…Bright red to red yellow
Nuclei………………………..…………………………………………….Light blue