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Alkaline phosphatase (NAP) staining solution (chemical staining method)

A technology of phosphatase and dyeing method, which is applied in the biological field, can solve the problems of inconspicuous positive staining, inhibition of enzyme activity, and low color sensitivity, and achieve the effect of improving color sensitivity, high sensitivity, and increasing positive staining

Active Publication Date: 2015-12-23
SHANGHAI SUNBIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, α-naphthol is unsubstituted naphthol, and the effective coupling with azo needs to add an excessive amount of diazonium salt, which may inhibit the enzyme activity, and its decomposition products have non-specific staining effect on cells, and every This experiment requires an ad-hoc preparation of incubation solution
In the later period, many scholars found substitutes for α-naphthol phosphate sodium salt, such as naphthol AS-BI phosphate sodium salt and naphthol AS-MX phosphate sodium salt. The application of this method for dyeing has the advantages of fast, simple, and easy repetition. However, there are always problems such as inconspicuous positive staining and low color sensitivity.

Method used

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  • Alkaline phosphatase (NAP) staining solution (chemical staining method)
  • Alkaline phosphatase (NAP) staining solution (chemical staining method)
  • Alkaline phosphatase (NAP) staining solution (chemical staining method)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Synthesis of N-(2-methyl-5-methoxy-4-biphenyl)-3-phosphate oxy-2-naphthylcarboxamide

[0049] Add 54g (0.29mol) of 2-hydroxy-3-naphthoic acid, 600ml of anhydrous xylene, and 57g (0.27mol) of 5-methyl-4-phenyl-o-anisidine into a 1L three-vessel with a serpentine reflux condenser In a glass bottle, stir in a water bath at 80°C for 10 minutes, add 0.09mol phosphorus trichloride, and reflux for 2 hours; collect the supernatant while it is hot, cool and precipitate the precipitate, and filter the filtrate; wash the precipitate with xylene and distilled water in turn; put the precipitate in In 3% HCl aqueous solution, heat and filter to obtain the filtrate, cool the filtrate again to precipitate the precipitate, wash the precipitate with hot distilled water, dry the precipitate, recrystallize and filter to obtain yellow 3-hydroxy-N-(2-methyl- 5-methoxy-4-biphenyl)-2-naphthylcarboxamide intermediate.

[0050] Dissolve 19.17g (0.05mol) of the above intermediate in 6...

Embodiment 2

[0051] Embodiment 2: composition and preparation of the kit of the present invention

[0052] Fixative: 10% formaldehyde methanol reagent, packed in 2.5ml / bottle;

[0053] Diazonium salt: solid purple B salt, divided into 30mg / bottle

[0054] Alkaline phosphatase substrate solution: 180mg N-(2-methyl-5-methoxy-4-biphenyl)-3-phosphateoxy-2-naphthylcarboxamide was dissolved in 3ml DMF, added to 600ml containing 0.1M Tris-HCl, 0.1M NaCl solution, adjust the pH value to 9.3, and pack in 30ml / bottle.

[0055] Hematoxylin counterstaining solution: 1.0g hematoxylin, 25ml absolute ethanol, 0.1g citric acid, 50ml glycerin, 5.0g aluminum potassium sulfate, 0.1g sodium iodate, 50ml distilled water.

[0056] Preparation method: dissolve hematoxylin in 25ml of absolute ethanol, when the hematoxylin is dissolved, add glycerin and shake the container; grind potassium aluminum sulfate and heat, then dissolve in distilled water, slowly add it to the above solution, And keep shaking, after b...

Embodiment 3

[0057] Embodiment 3: dyeing method of the present invention

[0058] (1) Fixation: Add dropwise 4°C pre-cooled fixative solution to the fresh smear for 30 seconds, wash with water and wait to dry;

[0059] (2) Staining: Put 1 bottle of diazonium salt reagent (30mg, 0.068mmol) into 1 bottle of alkaline phosphatase substrate solution (8.96mg, 0.019mmol) to completely dissolve and mix to form a working solution. Put it into the working solution, incubate at 37°C for 30 minutes, then rinse it with running water for 1-5 minutes, take it out and let it dry;

[0060] (3) Counterstaining with hematoxylin: add hematoxylin dropwise to the smear, counterstain for 1 to 2 minutes at room temperature, wash with water and wait until dry, and examine under the microscope.

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Abstract

The invention relates to the technical field of biology, and discloses a neutrophile granulocyte alkaline phosphatase staining kit and a staining method. The kit comprises an alkaline phosphatase substrate solution with the pH value being 9.2-9.8. The alkaline phosphatase substrate is N-(2-methyl-5-methoxyl-4-xenyl)-3-phosphoric acid oxygroup-2-naphthamide. The alkaline phosphatase substrate replaces existing naphthol AS-BI sodium phosphates, naphthol AS-MX sodium phosphates and the like for conducting NAP staining, chromophoric group xenyl is introduced in the chemical structure of the substrate, the reacting color intensity of naphthol produced by hydrolysis of the substrate by the alkaline phosphatase and tetrazonium salt is enhanced, and therefore the color display sensitivity and positive staining of the alkaline phosphatase are greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an alkaline phosphatase (NAP) staining solution (chemical staining method), namely a neutrophil alkaline phosphatase staining kit and a staining method. Background technique [0002] Alkaline phosphatase (NAP), mainly present in mature neutrophils, is an intracellular non-specific hydrolase that can hydrolyze various phosphates under alkaline conditions. As cells mature, the activity of NAP increases gradually. Under pathological conditions, different diseases and different stages of the disease can cause changes in the composition and content of NAP. Therefore, chemical staining as a rapid determination of alkaline phosphatase activity is often used as an auxiliary clinical diagnostic index for various diseases. The change of NAP activity has important clinical value for the identification and diagnosis of certain diseases: when suffering from chronic myelogenous leukemia (without ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
Inventor 谢永华朱美萍许付
Owner SHANGHAI SUNBIO TECH
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