Negative-staining electron microscopy (EM) and immuno-EM

For negative-staining EM, purified exosomes were fixed in 2% (v/v) paraformaldehyde for 5 min at room temperature. After fixation, 10 μg of the exosome suspensions was applied to formvar/carbon-coated grids (200 mesh) for 3 min and stained with 2% uranyl acetate. After excess uranyl acetate was removed with filter paper, the grids were examined under a transmission electron microscope (Hitachi H7600, Hitachi, Tokyo, Japan) at 80 kV. For immuno-EM, we used a modified whole-mount immuno-EM method²⁹. Briefly, purified exosomes were incubated with anti-PD-L1 (or PD-L1 isotype) antibody in blocking buffer (PBS containing 1% bovine serum albumin [BSA]) for 2 h. Then, 5 μg of the exosome suspensions was applied to formvar/carbon-coated nickel grids (200 mesh) for 3 min. We then washed the grids with five separate drops (50 µL, 10 min per drop) of PBS containing 0.1% BSA, transferred a drop of secondary antibody to the grids for 1 h (anti-mouse immunoglobulin G (IgG) conjugated to a 9–11 nm gold particle (1:100 in PBS containing 0.1% BSA), and repeated the washing procedure. Next, the grids were washed with two separate drops (50 µL) of distilled water. After negative staining with 2% uranyl acetate for 1 min, the specimens were viewed under a transmission electron microscope at 80 kV (Hitachi H-7600, Hitachi).