Acid Phosphatase for Glycol Methacrylate Sections Procedure:

Technovit Glycol Methacrylate: Technical Information and Help From Heraeus Kulzer.

  1. Incubate sections in the incubating medium at 37C 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 4C.
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 4C.
Sodium Nitrite Solution
  • Sodium Nitrite - 1 gm
  • Distilled Water - 25 ml
Prepare fresh and store at 4C.
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.


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