Ⅰ. Experimental steps of IHC staining in paraffin section

1.Paraffin section dewaxing to water(60 ℃ for 1 hour before paraffin staining.):
①Xylene Ⅰ、Ⅱ，10 minutes each.
②Gradient alcohol：100%， 2 minutes → 95% ， 2 minutes → 80% ， 2 minutes → 70% 2 minutes.
③Distilled water washing：5 minutes, 2 times (placed in the shaker).

2.Hydrogen peroxide blocking endogenous peroxidase：3% H²O²,room temperature 10 minutes (dark).

3.5 minutes, 2 times (placed in the shaker).

4.Antigen repair：Select the appropriate method according to the antigen to be detected.
Precautions for antigen repair:
①Organization can't do it.
②The selection of antigen repair methods depends on the antibody.
③This method is mainly used for 10% formalin fixed and paraffin embedded tissues.
④PBS buffer is needed during the process from antigen repair to dab color development.

5.5 minutes, 2 times (in a shaker).

6.Normal serum blocking：Remove the slice from the dye cylinder, wipe the moisture on the back of the slice and around the tissue on the front of the slice（Keep the tissue moist）. Treatment with normal goat or rabbit serum（Animal serum homologous with the second antibody）.37 ℃, 15 minutes.

7.Drop the first antibody：Remove the serum with filter paper, do not wash, directly drop the first antibody, 37 ℃ for 2 hours(it can also be placed in 4 ℃ refrigerator overnight).

8.PBS:5 minutes, 2 times (placed in the shaker).

9.he second antibody of biotinization by dropping，37℃，40 minutes.

10.PBS:5 minutes, 2 times (placed in the shaker).

11.Add three anti (SAB complex) drops, 37 ℃, 40 minutes.

12.PBS:5 minutes, 2 times (placed in the shaker).

13.DAB color development, microscopic observation, timely termination (water flushing termination).

14.Wash the tap water (fine water) thoroughly.

15.Hematoxylin re dyeing,room temperature, 30 seconds, tap water rinse.

16.Tap water rinse back to blue,15 minutes.

17.Gradient alcohol dehydration: 80%，2 minutes → 95%，2 minutes → 100%，2 次，5 minutes.

18.Xylene transparent:Ⅰ、Ⅱ，10 minutes each.

19.Seals:Canadian gum (or neutral gum) seal.

Ⅱ. IHC staining of cell slide

1.Take out the cell slide and fix it with cold acetone for 20 ~ 30 minutes.

2.Soak in distilled water for 5 minutes, twice.

3.Soak the drilling solution for 5 minutes.

4.Soak in distilled water for 5 minutes, twice.

5.Followed by step 6 of the previous experimental steps（Normal serum blocking）.

PS：Step 3 and step 4 are only used to detect intracellular antigen, not membrane antigen.

Ⅲ. IHC staining of frozen sections

1.The fresh tissue was immediately sliced in a cryostat (or stored at - 80 ℃), with a thickness of 5 ~ 6 μm.

2.The glass slide does not need to be bottomed. After mounting the slide, blow it dry with an electric hair dryer immediately.

3.If it is not dyed immediately, it can be sealed and stored at - 20 ℃.

4.Before dyeing, fix it with cold acetone at 4 ℃ for 10-20 minutes.

5.Wash PBS twice for 5 minutes each time (if necessary, use 0.1% sodium citrate + 0.1% Triton for drilling).

6.Inactivation of endogenous peroxidase by 3% H²O², 20 minutes, dark.

7.Soak in distilled water for 5 minutes, twice.

8.Connect to step 6 of the previous experiment.

Ⅳ.Pretreatment of slices

1.Soak the detergent for 30 minutes, wash and dry.

2.Wash solution (including strong acid, potassium permanganate, etc.) for 24 hours, wash and dry.

3.APES（1：50 Acetone solution）10-20 seconds.（notice：Do not use plastic containers and plastic chip holders.)

4.Pure acetone I, about 10 seconds. Pure acetone II, about 5 seconds.

5.Dry in air or oven, set aside.