8 1 REMOVAL of FORMALIN PIGMENT

PRINCIPLE
Formalin pigment may be present in tissue after long-term fixation in formalin with an acid pH. It is brown and usually extracellular, but may be present in macrophages. Short term fixation in neutral buffered formalin may prevent this artefact. It is easily removed with alcohol - ammonia solution.

REFERENCES
p 69 Cook H C, Manual of Histological Demonstration Techniques. p 243 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition.

REAGENTS
(A) ALCOHOL-AMMONIA SOLUTION 95 % alcohol 50 mL Concentrated ammonia (0.880) 15 mL Mix before use.

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Place in Alcohol-ammonia solution for 1 (one) hour.
3. Wash in water.
4. Stain using H&E or other technique.

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8 2 REMOVAL of MERCURY PIGMENT

PRINCIPLE
Mercuric chloride is found to a variable extent after fixation in mercuric chloride - containing fixatives, especially after long fixation. The granular black deposit can easily be removed by treatment in Lugols iodine and subsequent treatment with sodium thiosulphate.

REFERENCES
p 69 Cook H C, Manual of Histological Demonstration Techniques. p 243 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition.

REAGENTS
(A) LUGOLS IODINE Iodine 1.0 g Potassium iodide 2.0 g Distilled water 100 mL Alternatively,Verhoeff's solution C from Elastic stain may be used for 5 minutes.
(B) 5 % AQUEOUS SODIUM THIOSULPHATE Use from retic. stain.

PROCEDURE
1. Bring sections to water.
2. Place in Lugol's iodine for 15 minutes.
3. Wash in water.
4. Place in thiosulphate for 3 mins.
5. Wash in water.
6. Stain with H&E or other technique.

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8.3 DEMONSTRATION OF BILE
See 8.3.1 and 8.3.2. 8 3.1 DEMONSTRATION OF BILE AND HAEMATOIDIN

Bile pigment may be demonstrated by the following methods currently in use in this laboratory.

REFERENCE
p246 Bancroft & Stevens, Theory and Practice of Histological Techniques.

HAEMATOXYLIN AND EOSIN
Liver bile Golden brown
Gall bladder bile Gold
Haematoidin Gold

VAN GIESON METHOD (See 2.4)
Liver bile Bright green
Gall bladder bile Yellow
Haematoidin Yellow The green colour of liver bile by this method may be due to its high biliverdin content.

SIRIUS RED METHOD
Bile products Emerald green

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8.3.2 FOUCHET'S REACTION FOR BILE PIGMENTS
Van Gieson method preferred.

PRINCIPLE
In the presence of trichloracetic acid the bile pigments are oxidized by ferric chloride to form a mixture of green bilirubin and blue cholecyanin.

REFERENCES
p 77 Cook H C, Manual of Histological Demonstration Techniques. p 248 Bancroft JD & Stevens Theory and Practice of Histological Techniques, 2nd Edition

SPECIMEN
Standard paraffin sections 10% neutral buffered formalin fixation. Avoid mercury salts.

CONTROL
Gall bladder wall containing Bile pigment.
Note: Controls required:
1. Section not treated with Fouchet's reagent but counterstained with Van Gieson, i.e. Van Gieson only.
2. Section treated with Fouchet's but not counterstained with Van Gieson, i.e. Fouchet's Reagent only

REAGENTS
(A) FOUCHETS REAGENT 25 % trichloroacetic acid (aq) 25 mL 10 % ferric chloride (aq) [corrosive] 25 mL
(B) VAN GIESON'S SOLUTION (see Van Gieson's method 2.4) or SIRIUS RED STAIN (method 2.8)

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Treat with Fouchet's reagent for 5 minutes.
3. Rinse well in distilled water.
4. Counterstain with Van Gieson's solution for 2 minutes or Sirius Red Stain 30 minutes.
5. Rinse rapidly in water.
6. Dehydrate rapidly, Clear and mount.
Note: Steps 5 and 6 - the delicate red and yellow counterstaining is easily lost in water and alcoholic solutions.

RESULTS
Bile pigments Blue / green
Muscle Yellow
Collagen Red
 

8.4 DEMONSTRATION OF FERRIC IRON
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8.4.1 PERL'S PRUSSIAN BLUE FOR FERRIC IRON

PRINCIPLE
Ferric iron is released from any attachments to protein by treatment with dilute hydrochloric acid, which then reacts with a dilute solution of potassium ferrocyanide to produce an insoluble blue compound, ferric ferrocyanide (Prussian Blue).

REFERENCES
p 70 Cook H C, Manual of Histological Demonstration Techniques. p 258 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin sections 10% neutral buffered formalin fixation

CONTROL
Tissue containing iron salts. See next page for modification and procedure currently in use.

REAGENTS
(A) 2 % AQUEOUS POTASSIUM FERROCYANIDE
(B) 2 % AQUEOUS HYDROCHLORIC ACID
( C) 1 % AQUEOUS NEUTRAL RED

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Treat with 2% potassium ferrocyanide for 5 min. While waiting, mix equal volumes of 2% HCl and 2% ferrocyanide solution.
3. Drain off.
4. Flood slides with the mixture for 15 mins.
5. Wash well in water.
6. Counterstain with neutral red lightly.
7. Wash in water.
8. Dehydrate rapidly in alcohols.
9. Clear and mount.

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8.5 REMOVAL OF MELANIN PIGMENTS (BLEACH)

Melanins are brown-black pigments, which are bound to proteins and which are localised in cells in the cytoplasm. Melanins of various types are found in many sites including skin, eye and brain. Pathologically it is found in benign naevi and malignant melanoma.

PRINCIPLE
Melanin can be bleached by strong oxidising agents such as potassium permanganate.

REFERENCES
p 79 Cook H C, Manual of Histological Demonstration Techniques. p 255 Bancroft JD & Stevens Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin sections, formalin fixed

CONTROL
H&E without bleaching procedure, and H&E with bleaching procedure for each patient.

REAGENTS
(A) 0.25 % AQUEOUS POTASSIUM PERMANGANATE
(B) 5 % AQUEOUS OXALIC ACID

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Oxidise in permanganate for 30 minutes.
3. Water wash.
4. Bleach in oxalic acid until white.
5. Proceed staining as desired.

RESULTS
Skin melanin is bleached within 30 minutes Ocular melanin takes 2 - 4 hours.

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8.6 MASSON - FONTANA METHOD FOR MELANIN
(Masson, 1914; Fontana 1912)

PRINCIPLE
Melanin has the ability to reduce solutions of ammoniacal silver nitrate to metallic silver.

REFERENCES
p 64 Cook H C, Manual of Histological Demonstration Techniques. p 254 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin sections, formalin fixed

CONTROL
Tissue containing melanin pigment

REAGENTS
(A) PREPARATION OF SILVER SOLUTION To 20 mL of 10 % silver nitrate add concentrated ammonia drop by drop until the formed precipitate almost dissolves. A faint opalescence is evident when the end point is reached. If too much ammonia is added, 10% silver nitrate can be added drop by drop until the faint opalescence is obtained. To this silver solution add 20 mL of distilled water and filter. Store in a dark bottle. The solution will last for about four weeks in fridge 4øC.
(B) 5 % SODIUM THIOSULPHATE
(C) 1 % NEUTRAL RED

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Treat with silver solution a) 45 mins at 56øC to demonstrate melanin. b) at room temperature over-night in the dark to demonstrate Argentaffin.
3. Wash well in several changes of distilled water.
4. Treat with 5% sodium thiosulphate for 2 minutes.
5. Wash in tap water.
6. Counterstain in 1% neutral red (aqueous) for 3 minutes.
7. Wash in tap water.
8. Dehydrate through graded alcohols to Histoclear.
9. Mount in Safety-mount.

RESULTS
Melanin Black
Chromaffin Black
Some Lipofuscins Black
Nuclei Red
Argentaffin Black
Argyrophil cells do not stain

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8.7 SCHMORL'S REACTION FOR MELANIN
(Masson-Fontana method Preferred)

PRINCIPLE
Melanin has the ability to reduce ferricyanide to ferrocyanide, which in the presence of ferric ions forms Prussian Blue.

REFERENCES
p 252 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin or frozen sections

CONTROL
Melanin pigment

REAGENTS
(A) 1 % AQUEOUS FERRIC CHLORIDE
(B) 1 % AQUEOUS POTASSIUM FERRICYANIDE (freshly prepared) Working Solution To 30 mL of 1 % ferric chloride add 4 mL of 1 % potassium ferricyanide, then add 6 mLs of distilled water. Use within 30 minutes.
(C) VAN GIESON STAIN to counterstain (see method 2.4)

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Treat with Working Solution for 10 minutes.
3. Wash in water.
4. Counterstain with Van Gieson Stain for 2 minutes.
5. Dehydrate rapidly in alcohols.
6. Clear and Mount.

RESULTS
Melanin Dark blue
Argentaffin cells, Chromaffin cells, some Lipofuscins Blue
Nuclei Red

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8.8 MODIFIED VON KOSSA'S METHOD FOR CALCIUM
A modification of the classic von Kossa method of 1901.

PRINCIPLE
The classic method for the demonstration of calcium and certain other salts in tissues is that of von Kossa (1901). In the present context it is used as a negative stain for osteoid, the bone mineral being blackened by the deposition of silver and the osteoid remaining receptive to a contrasting stain.

REFERENCES
p 324 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard paraffin section

CONTROL
Calcium in tissue.

REAGENTS
(A) 1 % AQUEOUS SILVER NITRATE
(B) 2.5 % SODIUM THIOSULPHATE
(C) 1 % NEUTRAL RED OR VAN GIESON'S PICRO-FUCHSIN (See 2.4 )

PROCEDURE
1. Deparaffinize and hydrate to distilled water. Rinse well in distilled water
2. Place in Silver nitrate solution and expose to strong light - 10 to 60 minutes
3. Wash in 3 changes of distilled water.
4. Treat with Sodium thiosulphate for 5 minutes.
5. Wash well in distilled water.
6. Counterstain with neutral red 3 mins.
7. Dehydrate, clear and mount.

RESULTS
Mineralised bone Black
Osteoid Red

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8.9 TIMM'S COPPER STAIN - (MODIFIED)

PRINCIPLE
Copper is a normal constituent of many tissues in such small amounts that it cannot be demonstrated, however in Wilson's disease it slowly accumulates in certain organs, mainly in the liver and the basal ganglia region of the brain. Timm's copper stain is a silver technique where copper sulfide is converted to silver sulfide.

REFERENCES
Timm, Histochemie 2 : 332-341, 1961

SPECIMEN
Formalin or fresh tissue.

CONTROL
The following control procedures should be employed with contiguous sections:
1. Omit step 4 to stain all heavy metals.
2. Omit step 6.
3. Treat with 0.5 % potassium cyanide before developing to remove copper sulfide.
4. Stain with Perl's method for iron.

REAGENTS
(A) SOLUTION A 5.0 % Silver nitrate aqueous solution.
(B) SOLUTION B Hydroquinone 2.0 g Citric acid 5.0 g Distilled water 100.0 mL This solution can be stored at 4øC in the dark for one month.
(C) 0.1N HCl or 15% TRICHLORACETIC ACID

PROCEDURE
1. Deparaffinize in 2 changes of  Xylene and hydrate sections in water through graded alcohols.
2. Place in 0.5 % ammonium sulfide solution (aqueous) for 5 minutes.
3. Wash well in distilled water.
4. Treat sections with 0.1 N HCl for 2 to 3 minutes to remove iron & zinc sulfides (15 % trichloroacetic acid may also be used).
5. Rinse in distilled water.
6. Develop sections in a freshly filtered solution of 1 part A and 5 parts B. This converts copper sulfide to silver sulfide.
7. Rinse slides in distilled water.
8. Dehydrate through alcohols, clear in Histoclear, and mount in synthetic resin.

RESULTS
Copper deposits are stained black

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8.10 DEMONSTRATION OF URIC ACID AND URATES

PRINCIPLE
Uric acid and urates are breakdown products of purine metabolism which are further broken down and excreted in urine. In the disease gout, they are not broken down and accumulate in the body, becoming crystallised out into the tissues, particularly into the synovial membrane lining joint cavities. Uric acid and urates are soluble in water, so much of the material will be lost during normal aqueous fixation and subsequent processing. Staining with eosin only (no haematoxylin) provides limited contact with water.

SPECIMEN
95% alcohol is recommended as the most suitable fixative for the specific demonstration of urates. These specimens should be processed on a day cycle starting at absolute alcohol (station 5). Cut section at standard thickness. Avoid prolonged floating-out of section on waterbath.

REAGENTS
Eosin as used in linear stainer for routine H&E.

PROCEDURE
1. Floating-out of sections must not be prolonged.
2. Deparaffinise and hydrate section to 80 % alcohol (not to water).
3. Stain on linear stainer starting with Eosin.

RESULTS
Urates are birefringent and appear as pink needle-shaped crystals in Eosin only preparation.

NOTES
If a specimen is received in formalin, processed routinely and stained by H&E, urate crystals may appear blue to purple if numerous urate crystals were originally present. An alternative method of staining is the Gomori methenamine silver method. See Bancroft and Stevens, Theory and Practice of Histological Techniques, 4th ed p260.

RESULTS
Uric acid & urates black

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