IHC PROTOCOL

Immunochemistry, including immunohistochemistry (IHC) and immunocytochemistry (ICC), is the use of the principle of binding antigen and antibody with a high degree of specificity, through antigen-antibody binding and the presentation of the color reaction, to display the chemical composition of tissues or cells, and the qualitative, localization, or quantitative study of certain chemical components in the tissue section or cell specimen.

I. Dewaxing:

20min in xylene (Ⅰ), 20min in xylene (Ⅱ); 5min each in anhydrous ethanol (Ⅰ) and (Ⅱ); 5min in 95% ethanol; 5min in 80% ethanol; 5min in 60% ethanol; 3 times immersion in simple distilled water or deionized water, 1min each time.

II. Antigen repair:

l Place a specified amount of Citric Acid Repair Solution or Tris-EDTA Repair Solution in a heat-resistant container, place the discs in the container, then place the container in a microwave oven and heat for 10 minutes at power 5 or medium heat.

l Alternatively, a certain amount of restorative solution was added to a beaker and heated to boiling on an electric stove, and the slices were placed on a sectioning rack into the heated restorative solution and continued to boil for 15 minutes. After completion, they were left to cool naturally at room temperature for 30-40 minutes and then brought down to room temperature.

III. Immunohistochemical staining:

1. Sections were removed and washed by immersion in monodistilled or deionized water 3 times for 1 minute each;

2. After washing, the discs were immersed in a container of 3% hydrogen peroxide solution, covered, and left to soak for 10 minutes at room temperature under an airtight cover;

3. Sections were removed and washed by immersion in monodistilled or deionized water 3 times for 1 minute each;

4. Shake dry, wipe clean, add appropriate volume of 3% BSA dropwise, and close the wet box at room temperature for 1 hour;

5. Absorb excess liquid with absorbent paper (be careful not to dry the specimen), dropwise add appropriate amount of diluted primary antibody (primary antibody diluted with antibody diluent), incubate in a wet box at room temperature for 1 hour or overnight at 4℃;

6. 1xTBST rinsed 4-5 times for 30 seconds each;

7. Shake off, wipe clean, add appropriate amount of secondary antibody dropwise and incubate in a wet box at room temperature for 30 minutes;

8. 1xTBS washed 4-5 times for 30 seconds each;

9. Shake dry, wipe clean, drop appropriate amount of DAB solution, rinse 2-5 minutes later quickly with simple steam or deionized water; (DAB working solution ready for use).

10. Shake dry, wipe clean, add 1 drop of Mayor's Hematoxylin, restain for 1.5-2 minutes, soak in TBS solution for 5-10 minutes;

11. Sections were removed and washed by immersion in monodistilled or deionized water 3 times for 1 minute each;

12. Gradient dehydration in alcohol and xylene: 5 min in 60%, 80% and 95% alcohol, then 5 min each in anhydrous ethanol (I) and (II), followed by 5 min each in xylene (III) and (IV);

13. Sections were removed from xylene (IV), neutral rubber sealed, and examined microscopically.