7.1 BIELSCHOWSKY'S SILVER STAIN FOR AXONS (MODIFIED)

PRINCIPLE
This is a silver technique. Sections are first impregnated with silver nitrate and the deposit is intensified by means of a solution containing silver nitrate and a reducing substance - formalin.

REFERENCES
p 345 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition. SPECIMEN 5æ paraffin section 8 - 10 æ frozen sections

CONTROL
Nervous tissue Spinal cord

REAGENTS
(A) 20 % SILVER NITRATE SOLUTION (Caution: Silver nitrate is highly toxic and stains clothing/skin - wear gloves)
(B) 0.1 % AMMONIA SOLUTION
(C) DEVELOPER 10 % formalin 20 mL Distilled water 100 mL Concentrated nitric acid 1 drop (Caution: Highly toxic - burns skin) Citric acid 0.5 g
(D) 0.2% GOLD CHLORIDE (as for reticulin fibre stain method 2.5)
(E) 5 % SODIUM THIOSULPHATE
All solutions should be freshly prepared using clean glassware and distilled water.

EQUIPMENT
37øC water bath / oven

PROCEDURE
1. Deparaffinize and hydrate to distilled water.. Wash well in distilled water.
2. Place sections in 50 mL of 20% silver nitrate in the dark at 37øC for 15 mins. Keep this silver solution for stage 4.
3. Wash well in distilled water.
4. Add strong ammonia to the 50 mL of silver nitrate (from stage 2) until the initial precipitate disappears. Leave section in this solution for 10 minutes at 37øC.
5. Wash section in 0.1% ammonia.
6. Add 6 - 10 drops of developer solution to the silver hydroxide solution (from stage 4). Stain section for 3 - 5 minutes or until sections turn almost black.
7. Wash in ammoniated water and then distilled water.
8. Tone in 0.2% gold chloride for 1-2 minutes.
9. Fix in 5% sodium thiosulphate for 1 minute.
10. Wash, dehydrate, clear and mount.

RESULTS
Axons Black
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7.2 LUXOL FAST BLUE TECHNIQUE FOR MYELIN
(Kluuer and Barrera 1953)

PRINCIPLE
Luxol fast blue is one of the copper phthalocyanin dyes with a strong affinity for phospholipids and strong choline bases; hence presumably its affinity for myelin.

REFERENCES
p 162 Cook H C, Manual of Histological Demonstration Techniques.

SPECIMEN Standard paraffin section 8 - 10 æ frozen sections

CONTROL
Nervous tissue - cerebellum

REAGENTS
(A) LUXOL FAST BLUE SOLUTION Luxol fast blue 0.1 g 95 % alcohol 100 mL 10 % acetic acid 0.5 mL
(B) CRESYL FAST VIOLET SOLUTION 0.1 % (aq) NOTE: No CI number. Manufacture of Cresyl Fast Violet discontinued. Substitute Cresyl Violet Acetate (no CI number) Sigma C1791 or C5042.
(C) LITHIUM CARBONATE SOLUTION 0.05 % (aq)
(D) CRESYL VIOLET DIFFERENTIATOR 95 % alcohol 90 mL Chloroform 10 mL Glacial acetic acid 3 drops

EQUIPMENT
37øC water bath/oven

PROCEDURE
1. Deparaffinize and hydrate to 95 % alcohol.
2. Stain in Luxol Fast Blue overnight at 37øC.
3. Wash in 95 % alcohol, then in distilled water.
4. Commence differentiation by immersing sections in lithium carbonate solution for not more than 20 seconds.
5. Differentiate in 70 % alcohol until grey and white matter is clearly distinguished (30 - 60 seconds).
6. Rinse in distilled water and examine under microscope.
7. If differentiation not complete repeat steps 4 to 6 with reduced times.
8. Wash well in distilled water.
9. Stain in cresyl violet for 10 minutes.
10. Wash in distilled water.
11. Wash in 70 % alcohol.
12. Differentiate in cresyl violet differentiator 1 - 2 seconds.
13. Rinse in 95 % alcohol.
14. Rinse in absolute alcohol and clear in histolene.
15. Check differentiation under microscope to ensure only nuclei (and Nissl substance) are stained. Repeat steps 11 - 14 if necessary.
16. Mount.

RESULTS
Myelin, RBCs Blue Nuclei & Nissl substance Purple

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7.3 PAS - ORANGE G METHOD FOR ANTERIOR PITUITARY CELLS.
(Pearse, 1953)

PRINCIPLE
The pituitary gland has two main components, the adenohypophysis (anterior pituitary) and the neurohypophysis (posterior pituitary). The adenohypophysis is composed of solid masses of lightly packed epithelial cells which are divided into three groups on the basis of their staining characteristics.
1. Chromoplobe cells (approx. 50%) which have a scanty non-granular cytoplasm.
2. Eosinophil or acidophil cells (approx. 40%) found in clumps in the lateral wings of the anterior pituitary.
3. Basophil cells, which have blue staining (with H&E or aniline blue) granules in their cytoplasm. Numerous hormones are produced by the adenohypophysis, A.C.T.H., T.S.H., F.S.H. and L.H.

REFERENCES
p 370 Bancroft JD & Stevens, Theory and Practice of Histological Techniques, 2nd Edition.

SPECIMEN
Standard formalin fixed paraffin section

CONTROL
Normal pituitary gland

REAGENTS
(A) 1 % AQUEOUS PERIODIC ACID
(B) SCHIFF REAGENT (Use commercial product or refer to 6.1 for method to make Schiffs reagent)
(C) 2 % ORANGE G IN 5 % PHOSPHOTUNGSTIC ACID

PROCEDURE
1. Deparaffinize and hydrate to distilled water.
2. Oxidise with periodic acid for 8 minutes.
3. Rinse in water.
4. Treat with Schiff's reagent for 15 minutes.
5. Wash in running water for 10 minutes, this intensifies the colour reaction.
6. Stain the nuclei with an alum haematoxylin (no need to blue).
7. Stain in 2 % orange G in 5 % phosphotungstic acid 20 seconds.
8. Differentiate in tap water until macroscopically the section is pale yellow. Check microscopically that only the RBC's and acidophil cells are yellow.
9. Dehydrate, clear and mount in Safety Mount NOTES a) Other structures, e.g. collagen, will be PAS positive. If this obscures the delicate staining, the oxidation and Schiff's staining times should be reduced. b) This method is sometimes referred to as the Tri-PAS technique.

RESULTS
Basophil cells Magenta
Acidophil cells Yellow
Red blood cells Yellow
Nuclei Black
Chromophobes Pale blue - grey

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