Pd l1 e1j2j

The mIF analysis was performed using 4‐μm‐thick formalin‐fixed, paraffin‐embedded (FFPE) sections (Figure S2a). mIF was performed using an Opal seven‐color immunohistochemistry (IHC) kit (Akoya Biosciences). FFPE sections were fixed in 10% neutral‐buffered formalin (NBF) before paraffin block, then deparaffinized, rehydrated, and fixed one more time in 10% NBF buffer for 20 min before peroxide blocking. Antigen retrieval was performed using either citrate buffer or 0.1% sodium azide (pH 9) buffer (Nichirei Biosciences) and microwave treatment for 15 min. The slides were washed with TBST/0.5% Tween (three times, 2 min each) and incubated with 3% H₂O₂ for 10 min. Then, slides were incubated with the following primary antibodies: CD4 (4B12, Nichirei Biosciences, Ready to use/Opal 520), CD8 (C8/144B, Nichirei Biosciences, Ready to use/Opal 570), PD‐L1 (E1J2J, Cell Signaling Technology; 1:200/Opal 540), FoxP3 (236A/E7, Abcam; 1:100/Opal 620), pan‐CK (C11, Cell Signaling Technology; 1:500/Opal 690). CD8/CD4/PD‐L1/pan‐CK was used for membrane staining, FoxP3 for nuclear staining. Tyramide signal amplification solution was applied after the corresponding secondary HRP‐conjugated polymer for each mAb from the Opal seven‐color IHC kit. Nuclei were stained with spectral 4′, 6‐diamino‐2‐phenylindole (DAPI) and mounted using ProLong Gold Antifade Reagent (Cell Signaling Technology).