2.1 Immunohistochemical staining

The total number of cases in this study was 215. For each case, 9 consecutive sections were made for HE (Hematoxylin - Eosin) staining and immunohistochemical staining of 8 indexes including MLH1, MSH2, PMS2, MSH6, CD8, CD68, PDL1 and PD1. Because a few cases tissue is too little to the last 2-3 tissue section to complete the immunohistochemical evaluation. So some individual index staining is less than the total number of all cases. MLH1, MSH2, MSH6 and PMS2 evaluations were completed in all 215 cases, and 205 cases were evaluable for PD1, 202 cases for PDL1 [T]/PDL1, 205 cases for CD8, and 202 cases for CD68.

The primary antibodies used in this study (including anti-MLH1, -PMS2, -MSH2, -MSH6, -CD8, and -CD68 antibodies), DAB chromogenic solution, and UltraSensitive TMSP detection kit were purchased from Maixin (Maixin Inc., Fujian, China). Anti-PDL1 antibody ^28-8 (ab205921), anti-PD1 antibody [EPR4877(2)] (ab137132), and Universal HIER antigen retrieval reagent (10X) (ab208572) were purchased from Abcam (Cambridge, UK).

IHC was performed on 4 μm-thick tissue sections mainly as previously described ¹⁸ at room temperature (18-30 °C) according to manufacturer's instructions. The primary antibodies (anti-MLH1, -PMS2, -MSH2, -MSH6, -CD8, and -CD68 antibodies) were ready for use. Anti-PD1 and anti-PDL1 antibodies were diluted at 1:260 and 1:600, respectively. Phosphate buffer solution (PBS) was used as the negative control, and positive staining of normal gastric interstitial immune cells was the internal control (MLH1, PMS2, MSH2, and MSH6).