Visualizing Extracellular Vesicles using Electron Microscopy

For conventional electron microscopy, collagen I mix was prepared by mixing rat tail collagen I (final concentration 2.2 mg/mL) with 10 X PBS, 1 M HEPES, and H₂O, and the pH was neutralized by adding 1 M NaOH. Collagen gelation was conducted on coverslips for 10 min at RT. sEVs were added on top of the collagen-coated coverslips and incubated for 20 min at RT. After a washing step in phosphate buffer, coverslips were fixed with a mixture of 2% PFA and 0.2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), quenched with PBS/50 mM glycine, and processed for immunogold labeling using anti-CD63 or anti-CD9 antibody and PAG 10 nm as previously described [32 (link)]. Coverslips were then fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and processed for Epon (TAAB Laboratories Equipment) embedding and ultrathin sectioning. Sections were then contrasted with uranyl acetate and lead citrate, as previously described [32 (link)].
For EM analysis of EVs or of the pull-down of collagen fibrils incubated with EVs, EVs or pull down (prepared as described below) were spotted on formvar/carbon-coated copper/palladium grids, incubated for 20 min before fixation with PFA 2%/0.1 M phosphate buffer, and washing with water. Then, negative staining was performed using 0.4% uranyl acetate in methylcellulose.
The samples were analyzed with an 80 kV transmission electron microscope (Tecnai Spirit G2; Thermo Fischer, Eindhoven, The Netherlands) equipped with a 4k CCD camera (Quemesa, EMSIS, Münster, Germany).