Clone rpa t8

Tumors samples were frozen in optimum cutting temperature (Sakura Finetek, Torrance, CA, USA). Acetone-fixed cryosections (8 µm) were consecutively treated with 0.01% Triton X-100 for 15 min, 0.03% hyaluronidase for 15 min, FcR Block (Innovex Biosciences, Richmond, CA, USA) for 40 min, and Background Buster (Innovex Biosciences) for 20 min. The sections were then stained with primary antibodies diluted in PBS + 5% bovine serum albumin and 0.1% saponin for 1 h at room temperature, washed, and stained with the secondary antibodies at room temperature for 30 min. Nuclei were counterstained with DAPI (1 µg/ml) for 2 min. Primary antibodies included anti-hCD45 at 2 µg/ml (clone H130; BioLegend), FITC-conjugated anti-pan cytokeratin at 1:60 dilution (clone CK3-6H5; Miltenyi Biotec, Bergisch Gladbach, Germany), and AF647-conjugated anti-hCD8a at 1.25 µg/ml (clone RPA-T8; BioLegend). The secondary antibody included goat anti-mouse IgG1 AF568 at 1:2000 dilution (Thermo Fisher Scientific). For acquisition, data were acquired by sequential acquisition, and tile-scan imaging was performed on an SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).