Bz h3c

Paraffin‐embedded tumor sections were dewaxed in xylene and ethanol and autoclaved for 15 min in an antigen retrieval solution to retrieve their antigen epitopes, and endogenous peroxidase activity was blocked with 3% H₂O₂. Tissue sections were incubated overnight at 4°C with primary antibodies, including rabbit polyclonal anti‐CD3 (1:200 dilution; ab4055, Abcam, Cambridge, UK) for CD3⁺ T lymphocytes, mouse monoclonal anti‐CD8 (1:300 dilution; clone G10F5, BD Pharmingen, San Diego, CA, USA) for CD8⁺ T lymphocytes, anti‐CD4 (1:300 dilution; clone 10D6, Novocastra, Newcastle, UK) for CD4⁺ T lymphocytes, and anti‐FOXP3 (1:300 dilution; clone 10D6, Novocastra) for FOXP3⁺ T lymphocytes. The secondary antibody was incubated in a ready‐for‐use EnVision–peroxidase system (Dako Japan, Tokyo, Japan). Sections were incubated with horseradish peroxidase‐labeled polymer (EnVision1kit, Dako, Carpinteria, CA, USA) for 30 min at 25°C and incubated with 3,30‐diaminobenzidine tetrahydrochloride (applied as a 0.02% solution containing 0.005% H₂O₂ in 0.05 M Tris–HCl; pH 7.6) at 25°C for 5–15 min and counterstained with hematoxylin. We counted each positive lymphocyte in the invasive tumor margin using a BZ‐X700 digital microscope at a magnification of 200× (Keyence) using hybrid cell count software (BZ‐H3C; Keyence; Figure S1).