Carbon formvar coated copper grids

Platelet-derived EVs were fixed in 2% formaldehyde (Sigma-Aldrich, Saint Louis, MI, USA) solution. Fixed EVs were set on copper formvar-carbon-coated grids (Ted Pella, Redding, CA, USA) during 20 min and washed with PBS. Then, the grids with the EVs on it were incubated with 1% glutaraldehyde (Sigma-Aldrich) for 5 min and washed with deionized water. The grids were stained for 1 min with 2% uranyl acetate (Electron Microscopy Sciences Hatfield, PA, USA) and washed with PBS. Images were taken using a TEM-H600 (Hitachi, Tokyo, Japan) at 50 kV.

Procedures were performed essentially as described (Raposo et al., 1996 (link); Hurbain et al., 2017 (link)).
For transmission electron microscopy (TEM), sEV preparations were loaded on copper formvar/carbon coated grids (Ted Pella). Fixation was performed with 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), followed by a second fixation with PBS 1% glutaraldehyde in PBS. Samples were stained with 4% uranyl acetate in methylcellulose.
For immunolabeling electron microscopy (IEM), sEV preparations were loaded on grids and fixed with 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Immunodetection was performed with a mouse anti-human CD63 primary antibody (Abcam ab23792). Secondary incubation was next performed with a rabbit anti mouse Fc fragment (Dako Agilent Z0412). Grids were incubated with Protein A-Gold 10 nm (Cell Microscopy Center, Department of Cell Biology, Utrecht University). A second fixation step with 1% glutaraldehyde in PBS was performed. Grids were stained with uranyl acetate in methylcellulose.
All samples were examined with a Tecnai Spirit electron microscope (FEI, Eindhoven, The Netherlands), and digital acquisitions were made with a numeric 4k CCD camera (Quemesa, Olympus, Münster, Germany). Images were analyzed with iTEM software (EMSIS) and statistical studies were done with Prism-GraphPad Prism software (v8).