- 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
- 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
