Negative Staining of Extracellular Vesicles

The EVs were negatively stained using a protocol, as previously described [54 (link)]. Briefly, 4% paraformaldehyde (10 μL) was added to the EVs (10 μL), which incubated for 30 min. A carbon film grid (Electron Microscopy Sciences; Baltimore, MD, USA; CF200-Cu-25) was placed on the paraformaldehyde/EV droplet for 20 min and washed with PBS. Then, the grid was placed on 1% glutaraldehyde (50 μL) for 5 min and washed eight times with water. Finally, the grid was placed on uranyl acetate replacement stain (50 μL) for 10 min and left to dry for 10 min. The images were acquired on a JEOL JEM 2100 LaB6 TEM at 200 kV (40,000× magnification) using a digital camera (Gatan; Pleasanton, CA, USA).