Fei tecnai g2 spirit bio twin tem

In total, 5 µl EV preparation was fixed with 5 µl 4% paraformaldehyde at 4°C for 30 min and added onto formvar-coated copper grids to settle for 30 min. Subsequently, the samples were fixed with 1% glutaraldehyde for 5 min at 25°C, then stained with 2% uranyl oxalate for 5 min at 25°C and 1.8% methyl cellulose uranyl acetate for 10 min on ice in the dark, embedded in epoxy resin and polymerized at 35°C for 12 h, 45°C for 12 h and 60°C for 24 h. Residual liquid was removed from the grid with filter paper and the samples were visualized at 120 kV using the FEI Tecnai G2 Spirit Bio TWIN TEM (FEI; Thermo Fisher Scientific, Inc.). Images were acquired using an AMT 2k CCD camera (Advanced Microscopy Techniques, Corp.).