Alkaline phosphatase in cell tissues hydrolyzes the substrate (sodium naphthol a-monophosphate) in the action solution in an alkaline (PH 9.0-9.6) environment to produce a naphthol, which is then coupled with an azo salt to produce an insoluble sunlight-resistant dye (the color of the final product varies depending on the variety of azo salt used). This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.

Operation method

alkaline phosphatase staining assay

Principle

Alkaline phosphatase in cell tissues hydrolyzes the substrate (sodium naphthol a-monophosphate) in the action solution in an alkaline (pH 9.0-9.6) environment to produce a naphthol, which is then coupled with an azo salt to produce an insoluble sunlight-resistant dye (the color of the final product varies depending on the variety of azo salt used).

Materials and Instruments

Sodium Naphthol Phosphate Azide Salt Propylene Glycol

Move

I. Experimental reagents: Preparation of substrate solution: 35mg of sodium α-phosphononaphthol, 35mg of azide salt (FastGsrnet), 35ml of 0.05M propylene glycol (Propcmeliul) buffer, which can be filtered onto the smear directly after preparation.

Experimental methods:

l. Fix the smear in cold (0.5℃) 10% formalin methanol solution for 30 seconds.

2. Rinse with distilled water.

3. Add the substrate action solution dropwise on the smear, and act for 5-10 minutes at room temperature.

4. Rinse with tap water for 10 minutes.

5. Re-staining with 2% methylene green for 10 minutes.

6. Wash with water, air-drying, and sealing with glycerol.

III. red, can be used within the table scoring, according to the number of 100 neutrophils cytoplasmic NAP particles 0-4 + scoring, and then 100 cell scores will be added up, that is, the total integral value, note that only limited to mature band and polymorphonuclear neutrophils, not counting lymphocytes, eosinophils and basophils.



Note: · refers to the percentage of cell plasma accounted for by azo dye deposition.

Notes:

1. Smears should be fresh, appropriately thick and thin, and fixed in time.

2. Staining solution should be freshly prepared, and note that the pH value should be 9.4.

3. For each staining, it is best to make a blood smear from a patient with septicemia at the same time as a positive control, and to report the rate of positive cells and the integral value of the person undergoing the test and the control person at the same time.

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