Transmission Electron Microscopy of Extracellular Vesicles

Transmission electron microscopy characterization was performed as previously described (64 (link)). Briefly, isolated total pEVs were fixed with 4% glutaraldehyde (Electron Microscopy Services, Hatfield, PA) for 20 min at room temperature. A 10-μl droplet of glutaraldehyde-fixed pEVs was placed on Formvar-coated 300 mesh copper grid (Electron Microscopy Services). The sample was incubated for 1 min, followed by rinsing with distilled water for 1 min to ensure the removal of PBS salts. Excess liquid was blotted off with a Whatman filter. After rinsing, 50 μl of uranyl acetate solution was put on the grid and allowed to remain for 1 min. Excess liquid was removed, and the grids were viewed on a Hitachi H-7100 transmission electron microscope (Hitachi High Technologies) operating at 100 keV. Digital images were collected using an AMT Advantage 10 CCD camera system (Advanced Microscopy Techniques) and inspected using NIH ImageJ software (http://rsbweb.nih.gov/ij/).