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Acid Phosphatase for Glycol Methacrylate Sections Procedure

sprotocols

### Method: 1. 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: 1. 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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