### 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.