Acid phosphatase is found primarily in macrophages, localized in lysosomes.

Principle

The basic principle of the Acid Phosphatase Display Assay is that acid phosphatase reacts with the substrate sodium glycerophosphate (which contains a phosphate ester) at pH 5.0, causing the phosphate ester to hydrolyze to release a phosphate group, and the _PO3- binds to a lead salt to form a lead phosphate precipitate. The colorless lead phosphate then interacts with ammonium sulfide to form a yellowish-brown to brownish-black lead sulfide precipitate, thus indicating the presence and distribution of acid phosphatase within the cell.

Operation method

Acid phosphatase display assay (acid phosphatase display assay)

Principle

The basic principle of the Acid Phosphatase Display Assay is that acid phosphatase reacts with the substrate sodium glycerophosphate (which contains a phosphate ester) at pH 5.0, causing the phosphate ester to hydrolyze to release a phosphate group, and the PO3- binds to a lead salt to form a lead phosphate precipitate. The colorless lead phosphate then interacts with ammonium sulfide to form a yellowish-brown to brownish-black lead sulfide precipitate, thus indicating the presence and distribution of acid phosphatase within the cell.

Materials and Instruments

Equipment:
Thermostat, refrigerator, microscope, routine laboratory instruments, mouse peritoneal fluid smear.
Reagents:
① 6% starch broth
(Beef paste, 0.3 g; peptone, 1.0 g; sodium chloride, 0.5 g; soluble starch, 6.0 g; distilled water, 100 mL). Sterilize by boiling and store in refrigerator at 4 ℃. (Melt with warm water when using.)
Acid phosphatase working solution
(Lead nitrate, 25 mg; acetate buffer (0.05 mol ／ L), 22.5 mL; 3% sodium β-glycerophosphate, 2.5 mL). Add lead nitrate into the acetate buffer, stir to dissolve all of it, and then slowly drop 3% sodium β-glycerophosphate 2.5 mL, while stirring rapidly to prevent flocculation and affecting the experimental results.)
③Formaldehyde-calcium fixative
(40% formalin, 10 mL; 10% CaCl
2
(40% formalin, 10 mL; 10% CaCl2 (anhydrous) aqueous solution, 10 mL; distilled water, 80 mL).
④2% ammonium sulfide solution
⑤ Glycerol gelatin sealer

Move

The basic procedure of the acid phosphatase display assay can be divided into the following steps: (i) Smear of peritoneal fluid A. Take 1 mouse and inject 1 mL of 6% starch broth intraperitoneally every day for 3 days.B. On the third day, 3-4 h after the injection, 1 mL of saline was injected intraperitoneally, and 3 min later, the mice were executed by cervical dislocation, the abdominal cavity was dissected, and the intraperitoneal fluid was aspirated by a syringe without a needle.C. Put 1~2 drops of intraperitoneal fluid on a pre-cooled coverslip and immediately put it into the refrigerator (4 ℃) to let the cells spread out by themselves. 30 min later, remove the coverslip and blow it dry with cold air (it can be dried naturally at room temperature below 20 ℃).D. Put the cells into a small vat of acid phosphatase working solution and treat at 37 ℃ for 30 min.E. Wash the coverslips with distilled water for a few minutes, and put them on absorbent paper to remove the excess water.F. Transfer to formaldehyde-calcium fixative for 5 min.G. Wash with distilled water as in (5).H. Treat with 2% ammonium sulfide for 3-5 min .I. Wash with distilled water for a few moments.

J. Place 1 drop of glycerol gelatin sealer on the slide and seal the watered coverslip with the cell side down at the glycerol gelatin.

Caveat

1. Acid phosphatase is a soluble enzyme, cold fixation is better. After fixed by freezing or formaldehyde, the permeability of lysosomal membrane will be increased, and the substrate will be easier to enter into lysosome to be acted by the enzyme.

2. The incubation solution should be prepared before use, and added sequentially, until the lead nitrate is completely dissolved, and then add the substrate in small amounts one by one, and the incubation solution will become clear gradually.

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