Experimental principle
Immunocytochemistry is a technique for localizing antigens based on immunological principles by specifically binding antibodies to specific antigens. The antigen is mainly a macromolecule or a small molecule that binds to a macromolecule; an antibody is a gamma globulin secreted by a plasma cell against a specific antigen. If the antibody is bound to the label and then reacts with the antigen in the tissue, the site where the antigen is present in the tissue can be visualized under light or electron microscopy.
Commonly used markers are fluorescein and enzymes. Fluorescein-labeled is called immunofluorescent technique, and commonly used fluorescein is fluorescein isothiocyanate, rhodamine, and the like. Enzyme-labeled is called enzyme-labeled antibody method. The commonly used enzyme is horseradish peroxidase. The enzyme reacts with the substrate to form opaque deposits, indicating the presence of antigen. The part.
The method of binding an antibody to an antigen can be divided into a direct method and an indirect method. The direct method is to react a labeled antibody with an antigen to reveal a site where the antigen exists. The indirect rule is based on the primary reaction of the antibody antigen, and then the labeled secondary antibody is reacted with the primary antibody, thereby amplifying the primary reaction and showing enhancement.
Experimental procedure
1. Paraffin section 4-5μm;
2. Slice the wax to water;
3. 3% methanol hydrogen peroxide blocked room temperature for 10 minutes;
4. 0.01M (pH 7.2) phosphate buffer for 5 minutes and 3 times; antigen retrieval, add 0.01M (pH 6.0) citrate buffer in the microwave for 10 minutes, 3 minutes and 30 seconds, then 2 points and 1 point to 1 minute 30 seconds microwave maintenance.
5. After natural cooling, wash, add secondary antibody, non-immune serum of the same animal, and block at room temperature for 10 minutes;
6. Wash free, pour out the serum, and add the prepared primary antibody at 4 ° C overnight;
7. Wash 5 times 3 times;
8. Add biotin-labeled secondary antibody dropwise and incubate for 10 minutes at room temperature;
9. Wash 5 times 3 times;
10. Add streptavidin-peroxidase dropwise at room temperature for 10 minutes;
11. Wash 5 times 3 times;
12. Freshly prepared DAB solution develops for 3-10 minutes;
13. Washed with water, hematoxylin lightly stained the nucleus and differentiated. Tap water to rinse back, gradient alcohol dehydration, xylene transparent, neutral gum seal.
Abiraterone Acetate Intermediates
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Abiraterone Acetate,Abiraterone Acetate Intermediates,Cas 154229-19-3
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