Gb111337

Frozen slides were brought to room temperature, fixed in cold acetone for 10 min, then air dried. Samples were incubated in proteinase K working solution at 37°C for 25 min. Next, permeabilize working solution was used to cover objective tissue, then incubate at room temperature for 20 min. The slices were equilibrated at room temperature: The following kits and antibodies were used for staining: TUNEL Assay Kit (G1501, Servicebio, China), rabbit anti-KI67 antibody (1:200; GB121141, Servicebio, China), goat anti-CD3 antibody (1:200; GB111337, Servicebio, China), 488 anti-goat (1:400; GB25303, Servicebio, China) and Cy5 anti-rabbit (1:400; GB27303, Servicebio, China). The slices were washed three times with PBS (pH 7.4). Throwed away liquid slightly, then coverslip with anti-fade mounting medium. Images were acquired using an OLYMPUS laser scanning microscope.

Murine tumor tissues sections were fixed in 4% paraformaldehyde for 20 min at 20 °C and blocked for 10 min. Sections were then stained with anti-CD3 antibody (GB111337, Servicebio, Wuhan, China) overnight at 4 °C, Cy3 conjugated secondary antibody (GB21303, Servicebio, Wuhan, China) for 1 h, and DAPI (G1012, Servicebio, Wuhan, China) for 15 min in the dark. Images were captured with a fluorescence microscope.

The following antibodies were used in this research:
For flow cytometry analysis: anti-rat-CD3 (#201406, BioLegend), anti-rat-CD4 (#201509, BioLegend), anti-rat-CD8a (#201712, BioLegend), anti-rat-CD11b/c (#201817, BioLegend), anti-rat-granulocytes (#550002, BD Pharmingen), and anti-rat-CD68 (MCA341A700, Bio-Rad).
For immunohistochemistry: anti-myeloperoxidase (MPO) rabbit antibody (GB11224, Servicebio, 1:1,000), anti-CD68 rabbit antibody (GB11067, Servicebio, 1:500), anti-CD19 rabbit antibody (GB11061-1, Servicebio, 1:400), anti-CD3 rabbit antibody (GB111337, Servicebio, 1:1,000), anti-rat-endothelial-cell-antibody-1 (RECA-1, ab9774, Abcam, 1:200), and anti-CXCL1 (ab86436, Abcam, 1:200).
For immunocytochemistry: RECA-1 (ab9774, Abcam, 1:400), anti-CXCL1 (ab86436, Abcam, 1:100), and secondary antibodies (goat anti-mouse IgG Alexa Fluor^® 488, 1:200; goat anti-rabbit IgG Alexa Fluor 594^®, 1:200; ZSGB-BIO).
For Western blot: anti-p-p38 (#4511, Cell Signaling Technology, 1:1,000), anti-p38 (#8690, Cell Signaling Technology, 1:1,000), anti-p-p65 (#3033, Cell Signaling Technology, 1:1,000), anti-p65 (#8242, Cell Signaling Technology, 1:1,000), anti-GAPDH (#5174, Cell Signaling Technology, 1:2,000), anti-p47 (PA5-104250, Thermo Fisher Scientific, 1:2,000), and anti-p-p47 (PA5-99359, Thermo Fisher Scientific, 1:1,000).