Immunohistochemistry

Immunohistochemistry (IHC) is based on the principle of antigen-antibody specific reaction while proteins and other antigens in tissue sections are qualitatively and locatively studied. IHC can be divided into immunofluorescence method, immunoenzyme-labeled method and immunocolloidal gold technology according to the types of labeled substances. Among them, immunoenzyme-labeled method is the most commonly used technique at present.

The basic principle of immunoenzyme-labeled method is to first interact with the tissue with enzyme-labeled antibody, and then add the enzyme substrate to generate colored insoluble products or particles with a certain electron density. Through light or electron microscopy, various antigen components on the cell surface and inside the cell are studied. At present, peroxide-antiperoxidase (PAP) method, vitelloin-biotin-peroxidase complex (ABC) method, Streptomyces biotin-peroxidase link (SP) method are widely used in pathological diagnosis.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Wax melting: 72℃ wax melting 1-2 h.

2. Dewaxing: Slide immediately immersed in xylene for 5 min (three times).

3. Hydration: Anhydrous ethanol 3 min (three times), 95% ethanol 3 min, 90% ethanol 3 min, 75% ethanol 3 min, distilled water 5 min.

4. Blocking endogenous peroxidase activity: 3% H₂O₂ solution incubated slices at room temperature for 10 min and washed with PBS for 5 min (three times).

5. Antigen repair: Immersed in sodium citrate buffer, heated at 95-100℃ for 20 min, cooled to room temperature, TBST washed for 3 min (three times).

6. Sealing: Add 100 μL sealing liquid to each section and seal it at room temperature for 20-60 min.

7. Primary antibody incubation: Throw off the sealing solution and add primary antibody to each slice for overnight incubation at 4℃ or at room temperature for 45 min.

8. Washing: TBST 5 min (4 times).

9. Secondary antibody incubation: Add secondary antibody to each slice and incubate at room temperature for 30 min.

10. Washing: TBST 5 min (four times).

11. Dyeing: Drop DAB (now used) for color development for about 1 min (not too long), and TBST will terminate color development.

12. Redyeing: hematoxylin redyeing for about 30 s (not too long, can be repeatedly dyed), rinse with tap water for 10 min.

13. Dehydration: 75% ethanol 5 min, 95% ethanol 5 min, anhydrous ethanol 5 min (twice).

14. Sealing sheet: Use neutral resin to seal the sheet and observe the staining results with a microscope.

1, In order to meet the requirements of immunohistochemical technology, tissue fixation as fresh as possible;

2, Tissue dehydration must be thoroughly clean;

3. Hematoxylin restaining time needs to be explored, especially positive staining is also on the nucleus;

4. The color development time of DAB should be optimized to achieve obvious positive staining under the microscope but the background is not too deep;

5. Antibody incubation time and antibody concentration need to be explored. In particular, the incubation of primary antibodies is best spent overnight at 4 ℃;

6. Dewaxing and hydration of slices should be sufficient; When adding the reaction solution, the tissue should be fully covered; Shake the washing liquid dry before each addition, but prevent drying.