2.4.3. Analysis of Small Extracellular Vesicles by Scanning Electron Microscopy Imaging

Small EVs were fixed in a solution of 3.7% glutaraldehyde (Sigma–Aldrich, St. Luis, MO, USA) in PBS for 15 min, washed twice with PBS, and dehydrated through a series of ascending grades of ethanol (i.e., 40%, 60%, 80%, 96%–98%). Subsequently, samples were mounted on carbon adhesive stubs (Agar Scientifics, Stansted, UK) and left at room temperature for 24 h to obtain complete ethanol evaporation. Samples were gold-coated with a Balzers SCD 040 sputter coater (BAL–TEC AG, Balzers, Lichtenstein, Germany; thickness of gold layer: 40 nm) and analyzed at 132.21 K× magnification by a ZEISS EVO HD 15 Scanning Electron Microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) operating under high-vacuum at an accelerating voltage of 5 kV.
