Acid Phosphatase for Glycol Methacrylate Sections Procedure

• Protocol
• Discussion

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.
2. Wash in distilled water for 2 minutes.
3. Counterstain with Methyl Green for 5 minutes.
4. Wash in distilled water for 2 minutes.
5. Air dry and coverslip.
6. 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.
2. 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.
3. Substrate Solution
   • Naphtol AS-BI phosphatease, sodium salt (Sigma) - 40 mg
   • N.N-dimethylformamide - 4 ml
4. Pararosaniline Solution
   • Pararosaniline (C.I.# 42500) - 2 g
   • 2N HCl - 50 ml
   • Use heat to dissolve, filter when cool and store at 4°C.
5. Sodium Nitrite Solution
   • Sodium Nitrite - 1 gm
   • Distilled Water - 25 ml
   • Prepare fresh and store at 4°C.
6. Methyl Green
   • Methyl Green (C.I.# 42585) - 1 g
   • Phosphate/citrate buffer 0.1M pH 4.0 - 100 ml