Ix83 fv3000 confocal microscope

The murine PMs were cultured overnight on glass coverslips and then treated with lysosome inhibitors for 24 h. Then, the cells were coincubated with labeled αPD-L1 or TEV-bound αPD-L1 for another 2 h. After being washed three times with PBS, the cells were fixed with precooled methyl alcohol for 10 min at −20 °C and then permeabilized with 0.1% Triton X-100 for 10 min at RT. After the cells were blocked with 5% BSA and 3% goat serum in PBS, they were incubated with LAMP1 antibodies (Abcam, Cambridge, UK) overnight at 4 °C in blocking buffer. The following day, after three washes in PBS, the cells were incubated with DyLight 488-labeled secondary antibodies (Multi Sciences Biotech, Hangzhou, China) for 30 min at RT and washed in PBS. Finally, nuclei were stained with DAPI (Thermo Fisher Scientific). Liver and spleen tissues were embedded in Tissue-Tek™ CRYO-O.C.T. (Thermo Fisher Scientific) and processed to obtain 5 μm sections. Then, the tissue sections were stained with mouse F4/80 antibodies (Abcam) at 4 °C overnight followed by staining with DyLight 488-labeled secondary antibodies (Multi Sciences Biotech) for 1 h at 4 °C. The nuclei were stained with DAPI (Thermo Fisher Scientific) for 20 min at RT. The stained sections were imaged using an Olympus IX83-FV3000 confocal microscope (Olympus). Images were analyzed with ImageJ software (NIH).

HeLa cells were cultured overnight on glass coverslips and then treated with inhibitors or subjected to transfection. After being washed three times with PBS, the cells were fixed with ‐20°C prechilled methyl alcohol for 10 min and permeabilized with 0.1% Triton X‐100. After blocking with 5% BSA and 3% goat serum in PBS, the cells were incubated with primary antibodies (CD63, Invitrogen; HRS, Abcam; EEA1, abcam; LAMP1, abcam; Flotillin‐1, Abcam) overnight at 4°C in blocking buffer. The following day, after three washes in PBS, the cells were incubated with secondary antibodies (DyLight 488, DyLight 594, MultiSciences) for 30 min at RT, washed in PBS, and then mounted in antifade mounting medium with DAPI. The samples were imaged using an Olympus IX83‐FV3000 confocal microscope (Olympus). Images were analysed with ImageJ software.

The proximity ligation assay (PLA) were carried out using Duolink In Situ PLA reagents according to the manufacturer's instructions (Sigma) by using the primary antibodies (Coro1a, Abcam; Rab7, Abcam; GDI2, Abclonal; RILP, Proteintech). Samples were imaged using an Olympus IX83‐FV3000 confocal microscope (Olympus). Images were analysed with ImageJ software.