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        Alkaline Phosphatase for Glycol Methacrylate Sections

        互联网

        848

         

        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.

         

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