Alexa fluor 488 conjugated goat anti mouse secondary antibody

Staining for CD8 was done by incubation with the anti-CD8-α mouse mAb (dilution 1:200, Santa Cruz, USA) and Alexa Fluor^®488-conjugated goat anti-mouse secondary antibody (dilution 1:500, Absin, China). Anti-B7-2 mouse mAb conjugated to Alexa Fluor^®488 (dilution 1:200, Santa Cruz, USA) was used to detect CD86. Anti-CD206 mouse mAb conjugated to Alexa Fluor^®647 (dilution 1:200, Santa Cruz, USA) was used to detect CD206. Cell nuclei were stained with DAPI (dilution 1:5000, Biolegend, USA). The images were obtained with confocal microscopy.

The frozen tumors were cut into 10μm sections for immunofluorescence staining and confocal imaging. Staining for blood vessels was achieved by incubation with anti-CD31 mouse mAb conjugated to Alexa Fluor^®488 (dilution 1:200, Santa Cruz, USA). Anti-smooth muscle actin mouse mAb conjugated to Alexa Fluor^®647 (dilution 1:200, Santa Cruz, USA) was used for detection of α-SMA. Collagen I was detected with the anti-COL1A1 mouse mAb (dilution 1:200, Santa Cruz, USA) and Alexa Fluor^®488-conjugated goat anti-mouse secondary antibody (dilution 1:500, Absin, China). HIF-1α was detected with the anti-HIF-1α rabbit mAb (dilution 1:200, Cell Signaling Technology, USA) and Alexa Fluor^®488-conjugated goat anti-rabbit secondary antibody (dilution 1:500, Cell Signaling Technology, USA). PD1 was detected with the anti-PD1 rabbit mAb (dilution 1:200, Cell Signaling Technology, USA) and Alexa Fluor^®647-conjugated goat anti-rabbit secondary antibody (dilution 1:500, Cell Signaling Technology, USA). Cell nuclei were marked with DAPI staining (dilution 1:5000, Biolegend, USA). The images were captured at 20× magnification via confocal microscopic examination. The intensity values of the aim indicators were quantified with Image J software.