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Alkaline Phosphatase for Glycol Methacrylate Sections
Histology
scientificprotocols
authored about 6 years ago
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Procedure:
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:
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.
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