Immunohistochemistry

Freshly cut 4μm tissue sections were used for fluorescence multiplex immunohistochemistry (IHC) analysis. Antibodies against Ki67 (Cat. #DIA-670-P1, Dianova, mouse monoclonal antibody) and CD8 (Cat. #DIA-TC8, Oncodianova, mouse monoclonal antibody) were used to identify proliferating cytotoxic lymphocytes (Table 5). The OPAL dye kit (Cat. #OP7DS1001KT, PerkinElmer, Waltham, Massachusetts, United States) was used for antibody detection. The experimental procedure was according to the manufacturer’s instructions (PerkinElmer). Slides were initially boiled in a microwave oven (15 minutes at 100° C in pH9 buffer) for antigen retrieval. Antibodies to Ki67 and CD8 were combined with DAPI staining in each experiment. One circle of antibody staining included peroxidase blocking, application of the first primary (Ki67) antibody, detection with a secondary HRP-conjugated antibody, fluorescence dye detection, and removal of the bound antibodies by microwave treatment (15 minutes at 100° C). This cycle was repeated for the second primary (CD8) antibody. Slides were subsequently counterstained with diamidinoino-2-phenylindole (DAPI) and mounted in antifade solution.

(AR = antigen retrieval, RTU = Ready to use).