Bx61 fluorescence light microscope

Frozen tissue sections (8 μm thick) were fixed with 4% paraformaldehyde solution in PBS and used for the identification of CD8⁺ and CD4⁺ T cells using a rat anti‐CD8a mAb (1:150, 53‐6.7; BD Pharmingen) and a rat anti‐mouse CD4 (1:150, H129.19; BD Pharmingen), respectively. Anti‐interferon‐γ (IFN‐γ) Ab and rabbit polyclonal Ab (1:50; Abcam) were used to examine the localization of IFN‐γ. A rat anti‐CD31/PECAM‐1 mAb (1:150, MEC 13.3; BD Pharmingen), rabbit anti‐collagen IV polyclonal Ab (1:400; Abcam), and rabbit anti‐NG2 polyclonal Ab (1:150; Millipore) were used to detect endothelial cells, basement membrane, and pericytes, respectively. A rabbit anti‐TIM‐3 mAb (1:200, D3M9R; Cell Signaling Technology) was used to detect exhausted T cells. Alexa488‐ and Alexa594‐labeled secondary Abs (1:250; Invitrogen) were used for immunofluorescent detection. Nuclei were counter‐stained with DAPI (blue). In each slide, the number of positive cells was counted under a fluorescent microscope at 200× magnification. The pericyte coverage of the tumor vasculature was determined by double staining for CD31 and NG‐2. These images were acquired using an Olympus BX61 fluorescence light microscope.

The excised tumor tissues from the model mice were placed into OCT compound (Sakura Finetechnical Co.) and snap‐frozen. Frozen tissue sections (8 μm thick) were fixed with 4% paraformaldehyde solution in PBS and used for the identification of NK cells, CD8⁺ and CD4⁺ T cells, and T_regs using a goat anti‐mouse NKp46 (1:100; R&D Systems), rat anti‐CD8a mAb (1:150, 53–6.7; BD Pharmingen), rat anti‐mouse CD4 (1:150, H129.19; BD Pharmingen), and a rabbit anti‐Foxp3 polyclonal Ab (1:400 dilution; Novus Biologicals), respectively. To identify NKT cells, rat anti‐mouse CD3 (1:150, H129.19; BD Pharmingen), and a rabbit anti‐mouse CD49b polyclonal Ab (1:150 dilution, Thermo Fisher Scientific Inc.) were used. Alexa488‐ and Alexa594‐labeled secondary Abs (1:250; Thermo Fisher Scientific Inc.) were used for immunofluorescent detection. Nuclei were counter‐stained with DAPI (blue). In each slide, the number of positive cells was counted under a fluorescent microscope at ×200 magnification. These images were acquired using a BX61 fluorescence light microscope (Olympus).

Excised tumor tissues from AB1‐HA‐bearing mice were placed into Tissue‐Tek O.C.T. Compound (Sakura Finetechnical Co.) and snap‐frozen. Frozen tissue sections (8 μm thick) were fixed with 4% paraformaldehyde solution in phosphate buffered saline (PBS) and used for the identification of CD3⁺CD8⁺ T cells using a rabbit anti‐CD3 monoclonal antibody (1:150, SP7; Abcam) and a rat anti‐CD8a monoclonal antibody (1:150, 53–6.7; BD Pharmingen). Alexa 488‐labeled goat anti‐rabbit and Alexa 594‐labeled goat anti‐rat secondary antibody (1:250; Invitrogen) was used for immunofluorescent detection. Nuclei were counter‐stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (blue). In each slide, the number of double‐positive cells was counted under a fluorescent microscope at ×200 magnification. The images were acquired using an Olympus BX61 fluorescence light microscope (Olympus).