Acid Phosphatase for Glycol Methacrylate Sections Procedure

 

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.

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

Citation:

Gerrits, P. O. and Smid, L., "Staining Procedures for Tissues Embedded in 2-Hydroxyethyl Methacrylate", Heraeus Kulzer.