The intracellular esterase hydrolyzes the synthesized substrate naphthol ester (a naphthalene derivative) and releases naphthol, which is rapidly coupled with diazonium salts resulting in a brightly colored precipitate at the enzyme's active site. This experiment is from the Mudanjiang Medical College undergraduate 5-year laboratory guide for laboratory testing majors
Operation method
Specific esterase: chloroacetic acid AS-D naphthol esterase (NCE) staining assay
Principle
Intracellular esterases hydrolyze the synthesized substrate naphthol ester (a naphthalene derivative) and release naphthol, which is rapidly coupled to diazonium salts resulting in a brightly colored precipitate at the enzyme's active site.
Materials and Instruments
Phosphate buffer Hexaazotized new red Hematoxylin Ammonia washing solution Move I. Experimental reagents: Caveat l. Naphthol chloroacetate ester acid (NCE) reacts optimally between pH 7.O-7. 6， and it is not inhibited by sodium fluoride. 2. Strict adherence to the procedure, such as increasing the temperature and pH can lead to the decomposition of Naphthol Chloroacetate, resulting in a significant non-specific esterase reaction in the cell. 3. In addition to the method using new fachsin (new fachsin), some people choose to use fast garnet (GBC) or fast blue (fast blue, BBN). For more product details, please visit Aladdin Scientific website.
(i) Substrate mixing solution
(1) 0.067 mol/L phosphate buffer (PH 7.6)
(2) Hexaazolized new red (hexoliaed new fuchsin) solution (freshly prepared)
① Dissolve 1g of new fuchsin in 25ml of 2mol/L hydrochloric acid solution, the solution can be stored for a long time.
② 40g / L sodium nitrite solution, stored at 4 ℃, every week must be freshly prepared, before using the new volume of red solution and 40g / L sodium nitrite solution mixed, the reaction for 1min this solution is good for the six azotized new red
③Naphthol AS-D chloroacetate liquid: take naphthol AS-D chloroacetate (naphthol AS-D hcloroacetate) 5mg dissolved in N, N-dimethyl amide N, N-(dimeth for mamide) 2.5mg. dimeth for mamide) 2.5ml.
④ Naphthol AS-D hcloroacetate solution 2.5ml and 0.25ml of new red hexaazocarbonate added to 0.067mol/L phosphate buffer 47.5ml in the measurement of PH value and adjusted to 7.4 do not need to be can be used.
(ii) Recoloring solution: take 100ml of Harris hematoxylin (containing a small amount of alum) recoloring solution, add 4ml of glacial acetic acid and mix well.
(C) Dilute ammonia washing solution: add concentrated ammonia (28%) 0.2-0.3ml per 100ml distilled water and mix well.
II. Experimental operation:
1. Put the fixed blood or bone marrow slice into the substrate mixture, incubate at high temperature for 10min, and then rinse with running water.
2. Re-stain with re-staining solution for 10 min.
3. rinse the slide with dilute ammonia solution until the color changes from red to blue.
4. Rinse quickly with running water, dry in air and examine with an oil mirror.
Third, the results of the experiment to determine: a positive reaction for the red particles. Localized in the cytoplasm.