Immunohistochemistry
Tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin and sectioned with a thickness of 5 µm. Paraffin sections were dewaxed and hydrated with xylene and an ethanol gradient. After antigen repair with 0.01 mol/L citric acid buffer, the sections were tested with an immunohistochemistry kit (Abcam, Shanghai, China) using the following antibodies: anti-CD3 (Abcam, ab16669), anti-CD4 (Abcam, ab133616), anti-CD8 (Abcam, ab237709), anti-PD-1 (Abcam, ab52587) and anti-PD-L1 (Abcam, ab205921). They were diluted at 1:50 and incubated overnight at 4°C. After phosphate buffered saline (PBS) cleaning, biotin-labeled secondary antibody (concentration: 1:100) was added. After incubation at 37°C for 30 minutes, 3,3’-diaminobenzidine (DAB) color was developed for 15 minutes, then washed with tap water, stained with hematoxylin, sealed with xylene transparent resin, observed and photographed under a light microscope. According to the research methods in various studies, the scoring criteria for CD3-, CD4- and CD8-positive cells were from 0 to 3, of which 0 = no immune cell infiltration, 1 = only perivascular immune infiltration, 2 = perivascular + peritumoral and/or interstitial infiltration, and 3 = diffuse immune cell infiltration. Images showing immunohistochemical staining with score 1 to 3 are shown in Fig. 1A–C. The negative expression rate of PD-1/PD-L1 cell membrane or cytoplasm was <5%, and the positive expression rate was >5%. Five high power fields (HPF) were randomly selected from each section. [Figure 1 near here]