Tecnai 20 transmission

Samples were adsorbed for 1 min to a formvar film on a carbon-coated 400-mesh copper grid. The adsorbed samples were washed 3X in distilled water and negatively contrasted with 2% methylamine tungstate (Nano-W; Nanoprobes, Yaphank, NY, USA). Imaging was performed at 200 kV using a FEI Tecnai 20 transmission electron microscope (FEI Company, Hillsboro, OR, USA). Digital images of the specimens were acquired using an AMT Advantage XR 12 CCD camera (AMT, Danvers, MA, USA). TEM images were recorded at magnifications of 3,500 x to 19,000x.

EV samples were placed on glow discharged 200 mesh copper grids coated with formvar and carbon. After 1 min absorption, samples were dried, washed three times and stained with aqueous 2% uranyl acetate for 1 min. The stain was blotted dry from the grids with filter paper and samples were allowed to dry. Samples were then examined in a Tecnai 20 transmission electron microscope (FEI Company, Netherlands) at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp., USA).
Cryo-EM was used for direct visualization of extracellular vesicles. To prepare samples for cryo-EM study, lacey carbon EM grids were glow-discharged (30 s, 25 mA) in a Pelco EasiGlow system. An aliquot (3 μL) of the EV suspension in PBS was applied to the carbon side of an EM grid, which was then blotted for 3.0 s and plunge-frozen into the precooled liquid ethane. This procedure results in embedding the samples in a thin layer of amorphous ice to preserve them in their native state and to protect them from radiation damage. The samples were studied in a Talos Arctica (200 KeV, FEG) cryo-electron microscope.