Immunofluorescence

HMC-1 cells were fixed with 4% paraformaldehyde for half an hour. After washing with PBS 3 times, the cells were blocked with 5% BSA for 1 h. In a 4 °C refrigerator, the cells were incubated with PD-1 antibody (1:100, 18106-1-AP, Proteintech) for 1 h. After washing with TBST 3 times, the cells were incubated with cy3-conjugated goat anti-rabbit IgG (1:500, GB11013-1, Servicebio) for 1 h. Then, the cells were washed with TBST 3 times, and the nuclei were dyed with DAPI (G1012, Servicebio) for 10 min. After washing with TBST 3 times, the cells were resuscitated with anti-fluorescence quenching agent (G1401, Servicebio), the cell suspension was dropped onto a glass slide, and then pictures were taken with a laser confocal microscope. For sections of paraffin-embedded mouse tumors, we used chymase antibody (1:800, {"type":"entrez-nucleotide","attrs":{"text":"GB111085","term_id":"336598564","term_text":"GB111085"}}GB111085, Servicebio) to show mast cells and PD-1 antibody (1:2000, GB11338-1, Servicebio) to show the PD-1 receptor. We confirmed whether BMMCs were induced successfully by labeling with c-kit (1:800, GB11073-2, Servicebio).