Em 912 ab transmission electron microscope

Electron microscopy was performed on EV pellets stored at −80°C. For whole-mount analysis, EV suspensions (5 µL each) were incubated for 1 min on formvar and carbon-coated glow-discharged copper grids and subsequently stained with 2% uranyl acetate. Immunogold labelling was performed as described [22–24]. After blocking with 1% BSA in phosphate-buffered saline, grids were incubated with a 1:50 dilution of primary mouse anti-CD63 antibody (Thermo Fisher Scientific; TS63) in PBS containing 0.1% BSA for 1 h at room temperature. Extensive washes in the same buffer were followed by incubation with rabbit-anti-mouse antibody (Jackson ImmunoResearch) for 30 min, and after repeated washes 5-nm Protein A-gold (UMC Utrecht, the Netherlands) was applied for 1 h. After subsequent thorough washing, preparations were fixed using 1% glutaraldehyde in PBS, washed again and finally stained with 2% uranyl acetate. For controls, the primary antibody was omitted, resulting in complete loss of labelling (Supplementary figure S1). Preparations were inspected in an EM912 AB transmission electron microscope (Zeiss) at 120 kV under zero-loss conditions at slight underfocus. Images were recorded using a 2k x 2k slow-scan CCD camera (TRS, Germany) and the iTEM software package (Olympus-SIS).