Fitc or cy3 conjugated anti rabbit igg

For immunofluorescence staining, the tumor samples were fixed in 1% PFA, dehydrated overnight in 20% sucrose solution, and frozen (Leica). The frozen blocks were sectioned into 50 μm-thick slices, which were permeabilized with 0.3% PBS-T (Triton X-100 in PBS), and blocked with 5% normal goat serum in 0.1% PBS-T for 30 min at room temperature. Next, the samples were incubated overnight with the following primary antibodies: Anti-PD-L1 (rabbit, clone 28–8, Abcam), anti-CD8 (rat, clone 53–6.7, BD Pharmingen), anti-CD31 (hamster, clone 2H8, Millipore; rabbit, Abcam), anti-L-Kyn (mouse, clone 3D4-F2, ImmuSmol), anti-Granzyme B (rat, clone NGZB, Invitrogen), anti-Ki67 (rabbit, Abcam), or anti-Caspase3 (rabbit, R&D Systems). After several washes, the samples were incubated for 2 h at room temperature with the following secondary antibodies: FITC- or Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch), FITC- or Cy3-conjugated anti-rat IgG (Jackson ImmunoResearch), Cy3-conjugated anti-hamster IgG (Jackson ImmunoResearch), or FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch). Cell nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI, Invitrogen). Finally, samples were mounted with fluorescent mounting medium (DAKO), and images were acquired using a Zeiss LSM 880 microscope (Carl Zeiss).