SOP--Standard Operating Procedure
1. Paraffin sections were placed in a 65 ° C oven for 1 h, dewaxed to water, and washed three times with PBS for 5 min each.
2. The slices are placed in citrate buffer and the water bath is boiled, then timed for 30min.
3. After natural cooling, wash 3 times in PBS for 5 min each.
4. Place the sections in a 3% hydrogen peroxide solution and incubate for 10 min at room temperature to block endogenous peroxidase.
5. Wash 3 times in PBS for 5 min each time. After drying, block 5% BSA for 20 min (blocking charge).
6. Remove the BSA solution, and add 50 μl of the diluted primary antibody to each tissue to cover the tissue, and overnight at 4 ° C.
7. Wash 3 times in PBS for 5 min each.
8. Remove the PBS solution, add 50μl-100μl of the secondary antibody of the corresponding species to each section, and incubate at 4 ° C for 50min.
9. Wash 3 times in PBS for 5 min each.
10. Remove the PBS solution, add 50-100 μl of freshly prepared DAB solution to each section, and control the color development under the microscope.
11. After the color development is completed, rinse with distilled water or tap water, counterstain with hematoxylin, 1% hydrochloric acid alcohol differentiation (1s), rinse with tap water, return ammonia to blue, and rinse with running water.
12. The sections were subjected to a gradient of gradient alcohol (70-100%) for 10 min, dehydrated and dried, transparent xylene, and sealed with neutral gum.