Rabbit anti cd4

Mice in different groups were euthanized, and different tissues, including tumors, were excised and fixed in 4% paraformaldehyde. A part of tumors and DLNs were embedded, frozen, and sectioned at a thickness of 8 μm. All tissue section was treated according to the standard manufacturer’s instructions and stained with different primary antibodies: rabbit anti-CD11c (1:200), rat anti-F4/80 (1:200; Abcam), rabbit anti-CD11b (1:1000), rabbit anti–Ly-6G/Ly-6C, rabbit anti-CD4 (1:200), rabbit anti-Foxp3 (1:200), rabbit anti-CD206 (1:200), and rabbit anti–PD-L1 (1:200), followed by staining with fluorescently labeled secondary antibodies [Alexa Fluor 488–conjugated goat anti-rat IgG (H+L) (1:200; Thermo Fisher Scientific) and Alexa Fluor 633–conjugated goat anti-rabbit (H+L) (1:200; Thermo Fisher Scientific)]. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) containing mounting solution (DAPI Fluoromount-G, SouthernBiotech). All the slices were finally imaged with a confocal microscope (Leica, SP8). The other parts of tumor tissues and other organs were fixed in 4% paraformaldehyde, then embedded into paraffin, and subsequently sliced at a thickness of 5 μm. Slices were stained with hematoxylin and eosin (H&E), imaged by optical microscopy, and assessed by three independent pathologists.