Application suite x software 3

Tumor samples were fixed in 4% paraformaldehyde (Beyotime, China) and embedded in paraffin. The slides were incubated with primary antibodies at 4 °C overnight, then with HRP-conjugated goat anti-rabbit IgG secondary antibody (for IHC) or fluorescent-conjugated secondary antibody (for IF) at room temperature for 1 h in dark. The primary antibodies include: Ki67 (ab16667, Abcam), CD163 (ab182422, Abcam), CD4 (ab183685, Abcam), CD8 (ab217344, Abcam) and CCND1 (55506, Cell Signaling Technology). HRP-linked goat anti-rabbit IgG (ab6721, Abcam), Alexa Fluor^® 488-linked goat anti-rabbit IgG (ab150077, Abcam) and Alexa Fluor^® 555-linked goat anti-rabbit IgG (ab150078, Abcam) were used as secondary antibodies. Leica DM300 microscope was used to capture images. Leica Application Suite X software 3.0.0.15697 was used for quantitative analysis.

RAW 264.7 cells were cultured on slides in 6-cm dishes for 12 h, followed by co-culture with nanoparticles (5 mg/mL) for 6 h. The cells were incubated with stains (DAPI for nuclei staining, DIO for nanoparticles staining, and WGA for membrane staining) at 4 °C for 1 h in the dark. The stains included DAPI (DA002, Leagene Biotechnology, Shanghai, China), DIO (D4007, US Everbright®Inc., Suzhou, China), and WGA (MP6326-1MG, MKBio, Shanghai, China). A Leica DM300 microscope was used to capture the images. The Leica Application Suite X software 3.0.0.15697 was used for quantitative analysis.