Alkaline Phosphatase for Glycol Methacrylate Sections

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:

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

Solutions:

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.

Citation:

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