4 6 diamidino 2 phenylindole (dapi)

Free NVs, CR3022 NVs, B38 NVs, and CR3022/B38 NVs labeled with red fluorescent dye DIL (Abmole, USA) were separately co-incubated with 293 T-S cells to study the targeting of nanovesicles to 293 T-S cells. DIL labeling was performed as previously described. Briefly, 10 μg of nanovesicles were stained with 1 μL DIL at 37 °C for 30 min, before the mixture was centrifuged at 120,000 × g for 90 min to remove the free dye. The labeled nanovesicles were washed twice and then resuspended in 200 uL of PBS. Both 1 μg DIL labeled nanovesicles and 293 T-S cells were incubated in confocal culture dishes for 4 h, and the cells were washed twice with PBS and fixed for 30 min with 4% paraformaldehyde. The nucleus of 293 T-S cells was stained with the purple fluorescent dye DAPI (Abmole, USA). The binding of nanovesicles and 293 T-S cells after co-incubation was observed using a Zeiss LSM 880 confocal microscope.

Paraffin-embedded normal and tumor tissues were dewaxed, dehydrated, antigenically fixed, and sliced into 4 μm-thick sections for immunohistochemical staining. The sections were incubated with anti-human TREM2 antibody (Clone #237,920, 1:500, R&D) and anti-human CD206 antibody (#24,595, 1:1000, Cell Signaling Technology) overnight at 4 °C, followed by incubation with goat anti-rabbit secondary antibody (BOSTER), and finally stained with DAB (BOSTER) for visualization. DAPI (Abmole, USA) was used for staining of cell nuclei and blocking of sections prior to observation.