Granzyme b

The tissue samples were fixed in formalin and embedded in paraffin, and the paraffin blocks were sliced into 4 µm sections to conduct immunohistochemical (IHC) staining. Six serial sections of each paraffin-embedded tumor block were cut, one for H&E staining and five for IHC staining. The primary antibodies for TIL subsets were mouse monoclonal anti-human CD8 (DakoCytomation, Glostrup, Denmark; 1 : 100 dilution), granzyme B (Novocastra, Newcastle, UK; 1 : 100), OX40 (Novocastra; 1 : 30), FOXP3 (Abcam, Cambridge, UK; 1 : 50). The identification for the TLSs was the primary antibody to MECA-79 (1 : 300 dilution, Santa Cruz, California, USA), which recognizes sulfate-dependent carbohydrate epitopes of peripheral node addressin (PNAd) expressed in endothelial cells of HEVs. The IHC was performed as recommended by the manufacturer. The antigen retrieval was done by heat-induced epitope retrieval methods for 1 min and 30 s in citric buffer (pH 6.0). The detailed steps were the same as in our previous study [26 (link)]. Dako EnVision (DakoCytomation, Denmark) was used as the secondary antibody. Negative controls were done by omitting the primary antibodies. The tonsil tissue was used as positive controls.