Alkaline Phosphatase for Glycol Methacrylate Sections

• Protocol
• Discussion

Procedure:

1. Incubate sections in the incubating medium at room temperature for 1 - 3 hours. Two hours is sufficient in most cases.
2. Wash in distilled water for 2 minutes.
3. Counterstain with Nuclear Fast Red for 5 - 10 minutes.
4. Wash in distilled water for 2 minutes.
5. Air dry and coverslip.

Results:

1. Nuclei - red
2. Sites of enzyme activity - blue
3. To preserve the reaction product, selection of the right mounting (coverslipping) medium is important.

Solutions:

1. Incubating Medium
   • Naphtol AS-MX phosphate, di-sodium salt (Sigma) - 5 mg
   • N,N - dimethylformamide - 0.25 ml
   • Fast Blue BB (Sigma) - 30 mg
   • Distilled Water - 25 ml
   • Buffer Solution - 25 ml
   • 10% Magnesium Sulfate Solution. - 2 drops
   • Prepare fresh, shake well and filter before use.
2. Buffer Solution
   • 0.2M Tris (hydroxymethyl)-aminomethane - 2.4 gm
   • Distilled water - 100 ml
   • Adjust the pH of the buffer to 8.9 with dilute HCl and store at 4°C.
3. Nuclear Fast Red
   • To 0.2 gm of Nuclear Fast Red add 200 ml of boiling 0.5% aluminum sulfate solution.
   • Keep boiling for 5 - 10 minutes
   • Allow to cool and filter before use.