### Procedure:

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

### Results:

1. Nuclei - red
- Sites of enzyme activity - blue
- 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.
- 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.
- 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.