2.8. Multiplex immunohistochemistry (mIHC)

The tissue microarray slide containing human 96 GC tissues (Xinchao Biotechnology Co., Ltd.) was performed with mIHC. A total of 4 antibodies including anti‐MFSD2A (1:500, ab117618, Abcam), anti‐TGFβ1 (1:400, ab215715, Abcam), anti‐CD8 (1:600, 85336S, Cell Signaling Technology, Beverly, MA, USA) and anti‐Ki67 (1:600, 9449S, Cell Signaling Technology) were used. Briefly, the tissue microarray slide was dewaxed and hydrated and endoperoxidase activity was eliminated with 3% H₂O₂. After the blockade, the slide was incubated for 30 min with the primary antibody and was treated with HRP‐labeled secondary antibody (Perkin Elmer, MA, USA) for 10min. The staining process was repeated sequentially for each primary antibody. After the final staining round, 1 × 4′,6‐diamidino‐2‐phenylindole (DAPI; PerkinElmer) working solution was added dropwise on the glass slide, incubated for 5 min at room temperature, dipped with 1 × TBST (Sangon Biotech), and cover slipped with anti‐fluorescence quenching mounting medium (Sangon Biotech). MFSD2A, TGFβ1, and CD8 positive cell proportion scores were calculated by HALO v3.3.2541.301 (Indica Lab, Albuquerque, USA).