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Acid Phosphatase for Glycol Methacrylate Sections Procedure
Histology
scientificprotocols
authored over 6 years ago
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Method:
Incubate sections in the incubating medium at 37°C for 5 - 12 hours. Long incubation periods are needed to get significantly visible reaction product.
Wash in distilled water for 2 minutes.
Counterstain with Methyl Green for 5 minutes.
Wash in distilled water for 2 minutes.
Air dry and coverslip.
Results:
Nuclei - dark green
Cytoplasm - light green
Sites of enzyme activity - red
Solutions:
Incubating Medium
Combine 20 ml of buffer solution, 48 ml of distilled water and 4 ml. of substrate solution.
Combine 3.2 ml of pararosaniline solution with 3.2 ml of sodium nitrite solution. Mix for 1 minute.
Add the second solution to the first
Adjust pH to 5.
Buffer Solution
Anhydrous sodium acetate - 5.9 g
Sodium barbiturate - 14.7 g
Distilled water (boiled) - 500 ml
Do not adjust the pH of the buffer and store at 4°C.
Substrate Solution
Naphtol AS-BI phosphatease, sodium salt (Sigma) - 40 mg
N.N-dimethylformamide - 4 ml
Pararosaniline Solution
Pararosaniline (C.I.# 42500) - 2 g
2N HCl - 50 ml
Use heat to dissolve, filter when cool and store at 4°C.
Sodium Nitrite Solution
Sodium Nitrite - 1 gm
Distilled Water - 25 ml
Prepare fresh and store at 4°C.
Methyl Green
Methyl Green (C.I.# 42585) - 1 g
Phosphate/citrate buffer 0.1M pH 4.0 - 100 ml
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