Pe anti mouse cd8a antibody

Anti-Rabbit SMYD2 (#9734), Anti-Mouse Lamin A/C (#4777), Anti-Rabbit RIG-I (#3743), Anti-Rabbit Phospho-IRF-3 (Ser396) (#4764), Anti-Rabbit TBK1 (#3013), Anti-Rabbit Phospho-TBK1/NAK (Ser172) (#5483), Anti-Rabbit MAVS (#4983), Anti-Rabbit NF-kappa-B p65 (#4764), Anti-Rabbit Phospho-NF-kappa-B p65 (Ser536) (#3033), Anti-Mouse Phospho-I-kappa-B-alpha (Ser32) (#2859), Anti-Rabbit IKK-alpha (#2682), Anti-Rabbit IKK-beta (#2684), Phospho-IKK-alpha (Ser176)/IKK-beta (Ser177) (#2078) and Anti-rabbit IgG-488 (#4412) were from Cell Signaling Technology. Anti-PP1-alpha (sc-7482) was from Santa Cruz. APC anti-mouse CD11c (117310) was from Biolegend. Anti-Rabbit PP2-alpha (610555), Percp-Rat Anti-Mouse CD4 (553052), FITC-Rat Anti-Mouse CD49b (553857) and PE Rat Anti-CD11b (553311) were from BD PharMingen. PE anti-mouse CD8a Antibody (100708), APC anti-mouse CD11c Antibody (117310), PerCP/Cy5.5 anti-mouse CD19 Antibody (115534), FITC anti-mouse Ly-6G (127606), FITC anti-mouse CD3 (100204), anti-mouse F/480 FITC (123107) and PE anti-mouse I-A/I-E (107608) were from Biolegend. Recombinant Mouse GM-CSF (415-ML-050) and Recombinant Mouse IL-4 Protein (404-ML-01M) were from R&D Systems. Murine M-CSF (315-02-1000) was from PeproTech. LYY507 was donated by the University of Oxford. Camptothecin (#13637) was from Cell Signaling Technology. ³H SAM (NET155H001MC) was from PerkinElmer.

Splenic cells were obtained from the spleen samples by passing them through 100-mesh cell filtration sieves. The cells were then lysed with ammonium-chloride-potassium (ACK) lysis buffer at 4°C for 5 min. Next, 1 × 10⁶ splenic lymphocytes in phosphate-buffered saline (PBS) were prepared and stained with FITC anti-mouse CD3 antibody, APC anti-mouse CD4 antibody, and PE anti-mouse CD8a antibody (100204, 100412, and 100708, respectively; Biolegend) at a 1:250 dilution for 30 min in the dark at 4°C. The stained samples were analyzed using a BD FACSVerse flow cytometer, and the data were collected and analyzed using Flowjo 10 software.

Tumours were harvested from the mice and frozen in the cutting medium before sectioning via a cryotome. Before staining, the tissues were incubated with 1% BSA to block the nonspecific binding. For blood vessel staining, 10 μg mL⁻¹ of anti-mouse CD31 (R&D Systems, catalogue no. AF3628, goat IgG) antibody was incubated with the slides overnight, and the donkey anti-goat IgG (Invitrogen, catalogue no. 2044862, AF568-conjugated) secondary antibody was added after three times PBS washing, the nuclei were stained with Hoechst 33342. For platelet staining, 10 μg mL⁻¹ of anti-mouse CD62P (R&D Systems, catalogue no. AF737, goat IgG) were used. For the T cells staining, 20 μg mL⁻¹ PE anti-mouse CD8a antibody (Biolegend, cat no. 100708, clone: 53-6.7) and Alexa Fluor® 488 anti-mouse CD4 antibody (Biolegend, cat no. 100425, clone: GK1.5) was used to stain the tumour slices overnight at 4 °C. The tumour cell apoptosis was analysed with TUNEL. The stained slides were measured on a Zeiss LSM880 confocal laser scanning microscope and analysed with Zen 2 and Image J software.