Non-specific esterase staining assay

Summary

Nonspecific esterase (Nonspecific, esteras. NSE) staining method can be divided into three categories: neutral non-specific esterase, acidic non-specific esterase and alkaline non-specific esterase staining; non-specific esterase and its isoenzymes of blood cells are many kinds of enzymes, all the enzymes that act on short-chain fatty acids are called non-specific esterase. Neutral non-specific esterase staining method can be divided into three kinds due to different substrates: α-acetate naphthol esterase staining method, acetic acid and other AS acid enzyme staining method and acetate naphthol AS-D esterase staining method, and now we introduce α-acetate naphthol esterase (α-NAE). This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.

Operation method

Non-specific esterase staining assay

Principle

NAE hydrolyzes α-naphthol acetate to yield α-naphthol, which is then coupled to a diazonium salt to produce an insoluble colored precipitate located in the cytoplasm.

Materials and Instruments

Phosphate buffer α-naphthol acetate diazonium salt Methyl green water harbor pie

Move

I. Experimental reagents:

1. Action solution: l/20mol / L phosphate buffer (PH7.4) 40mg plus 10g / L α-acetate naphthol (50% acetone as a solvent) 0.8ml, vibrate fully until most of the turbidity initially produced disappears, add diazonium salt (Firm Blue B or other species can be) 40mg of vibration, filtration and then use.

2. 1g / L methyl green water Hongkong Pai

II. Experimental operations:

l. Fixed fresh smear in 10% formaldehyde saline fixed 5min

2. Rinse with running water for 5min

3. Add to 37℃ solution for 1 hour.

4. Wash with water

5. lg/L methyl green aqueous solution restaining 5min

6. Wash with water, dry and examine by microscope.

NaF inhibition test: the method is the same as above, in 40 ml of the action of the liquid add sodium fluoride (NaF) 6mg (per milliliter of the action of the liquid add NaF-l.5mg), if the positive obviously weakened or negative, showing that the NaF inhibition by the NaF inhibition of the best can be calculated as the NaF inhibition rate, the formula is:

(NSE integral value after adding NaF / NSE integral value without adding NaF) × 100% Where the inhibition rate is >50%, it suggests that it is indeed inhibited by NaF. IV. Experimental results: gray-black or brown-black diffuse or granular precipitation in the cytoplasm is positive, no precipitation in the cytoplasm is negative.

Common Problems

1. The specimen must be fresh and should be stained within two days of collection, otherwise the enzyme activity is significantly reduced. If stored in the refrigerator, the storage time can be extended appropriately.

2. The choice of diazonium salt, solid blue B, solid blue RR and solid black B diazonium salt staining is preferred.

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