acid phosphatase

Sections 5-10, micron unfixed, or cold formol calcium/gum sucrose fixed Cryostat sections.

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Stock solutionsA) Pararosanilin-HCL Stock

• Pararosanilin 1g
• Distilled water 20ml
• HCl Conc 5ml

The pararosanilin is dissolved in the distilled water and the hydrochloric acid is added. The solution is heated gently, cooled, filtered and stored in aliquots in a refrigerator.

B) Sodium Nitrite

• Sodium nitrite 2g
• Distilled water 50ml

This solution has to be prepared Fresh or made into 0.4ml aliquots and stored in the deep freeze.

C) Veronal-Acetate Buffer Stock

• Sodium Acetate (3H20) 3.88g
• Sodium Barbitone 5.88g
• Distilled water 200ml

D) Naphthol ASB1 Phosphate Stock

• Naphthol ASB1 Phosphate 50mg
• Dimethyl formamide 5ml

This is put into 0.5ml aliquots and stored in the deep freeze.

Preparation of incubating solution

• Pararosanilin - HCL (A) 0.4ml
• Sodium nitrite (40mg/ml) (B) 0.4ml

Add the pararosanilin drop by drop to the thawed sodium nitrite, shaking well after each addition until the solution is corn-coloured (leave to stand for at least 30 seconds).

• Naphthol ASB1 phosphate (D) 0.5ml
• Veronal Acetate Buffer Stock (C) 2.5ml
• Distilled water 6.5ml

Mix these together well and then add the pararosanilin/sodium nitrite solution. Adjust the pH to 4.7-5.0, filter and use immediately.

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Method

1. Incubate sections for 10-60 minutes at 37C
2. Wash well in distilled and then tap water
3. Counterstain, with 2% Methyl green for 15-30 seconds (haematoxylin can be used as an alternative)
4. Wash
5. Dehydrate, clear . Mount sections in DPX

Results

• Acid phosphatase activity - Red