Z0412

Procedures were performed essentially as described [25 (link),32 (link),33 (link)]. Immunodetection was carried out with the following primary antibodies: mouse anti-human CD63 (Abcam ab23792), mouse anti-human CD9, or mouse anti-human CD81 (both from Dr E. Rubinstein, Université Paris-Sud, Institut André Lwoff, Villejuif, France). Secondary incubation was performed with a rabbit anti mouse Fc fragment (Dako Agilent Z0412), then grids were incubated with Protein A-Gold 10 nm (Cell Microscopy Center, Department of Cell Biology, Utrecht University). All samples were observed 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 analysed with iTEM software (EMSIS) and statistical studies were done with GraphPad Prism software (v8).

A drop (5μl) of _SEVs (isolated by UC) suspended in PBS was deposited on formvar-carbon-coated electron microscopy grids for 20 min at room temperature, fixed with 2% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4), for 20 min at room temperature, and post fixed with 1% glutaraldehyde in PBS for 5 min at room temperature. Grids containing sEV were then washed and then blocked for 5 min at room temperature in blocking buffer (PBS, 1% BSA). sEVs were then immunolabelled with a mouse anti-human CD63 primary antibody (Abcam ab23792) diluted in blocking solution for 1 hour at room temperature, washed with PBS, 0,1% BSA, incubated with a rabbit antibody against mouse Fc fragment (Dako Agilent Z0412) in PBS 0,1% BSA for 20 min at room temperature. The preparations were then immunogold labeled with protein-A gold-conjugates (10 nm; Cell Microscopy Center, Department of Cell Biology, Utrecht University). Grids were analyzed on a Tecnai Spirit G2 electron microscope (Thermo Fischer Scientific) and digital acquisitions were made with a 4k CCD camera (Quemesa, Soft Imaging System). Images were analyzed with iTEM software (iTEM CE Olympus serie) and data with Prism-GraphPad Prims software (v8)⁶⁷ (link).

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).