Anti mouse cd31

Brains were fixed in 10% paraformaldehyde for 2 days then in 30% sucrose for 2 days. Brain sections (20 μm) were obtained on Leica cryostat (− 20 °C) then blocked with 5% goat serum staining buffer and stained with anti-mouse-CD31 (R&D systems, Minnesota, USA) overnight, then washed with PBS and stained with FITC-conjugated goat-anti-rabbit (Jackson Immunoresearch, Inc. Pennsylvania, USA) for 2 h. Brain sections were washed with PBS and mounted on glass slides using prolong gold anti-fade reagent (Life technologies, California, USA). The slides were photographed with a Nikon A1Rsi Confocal microscope (Nikon, Japan) using the Nis Elements 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.

Formalin‐fixed paraffin‐embedded tissue sections were dewaxed, and antigens were retrieved after 10 minutes of boiling in citrate buffer, pH 6 (Dako). Slides were stained with 10 μg/ml of biotinylated DVD antibodies for 1 hour at room temperature and were visualized with streptavidin–HRP complex using 3,3′‐diaminobenzidine chromogen (Dako). Rabbit anti–ICAM‐1 IgG (Abcam) was detected with HRP‐conjugated goat anti‐rabbit IgG (Jackson Immunotools). Mouse anti–von Willebrand factor (Dako) and mouse anti‐CD31 (R&D Systems) were used to identify human vascular endothelial cells, which were revealed with HRP‐conjugated goat anti‐mouse IgG (Santa Cruz Biotechnology). Sections were counterstained with hematoxylin, mounted with Depex mounting medium (Dako), and acquired with a CellSens imaging system (Olympus).