Uranyl oxalate

Aliquotes of concentrated EV samples were first fixed by mixing the samples 1:1 with 4% paraformaldehyde in DPBS. Fixed samples were transported on dry ice to the Polish Academy of Sciences in Olsztyn, Poland, for transmission electron microscopy (TEM) imaging. Fixed samples were contrasted in uranyl oxalate solution consisting of 4% uranyl acetate (Polysciences, Warrington, PA, USA) and 0.15 M oxalic acid (Sigma-Aldrich, Schnelldorf, Germany). The samples were subsequently embedded in a mixture of methylcellulose (Sigma-Aldrich, Schnelldorf, Germany) and uranyl acetate (Polysciences, Warrington, PA, USA). Embedded samples were imaged with a JEM 1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 80 kV, and digital images were acquired with numeric Morada TEM CCD camera, (Olympus, Hamburg, Germany).

Fractions 6-9 (800 µL) of isolated EVs on SEC were pooled and subsequently concentrated to 150µl using Amicon^® Ultra 2 centrifugal filter units (10 kDa) (Merck Millipore Ltd.). A previously described method [39 (link)] was followed for transmission electron microscopy (TEM) analysis. A droplet from the purified EV samples was deposited on Formvar-carbon-coated 200 mesh copper grids (Agar Scientific, Essex, UK) and allowed to absorb for 20 min. The sample was fixed on a grid in 2% paraformaldehyde (Sigma-Aldrich) and 1% glutaraldehyde (Polysciences, Warrington, PA, USA), contrasted in uranyl oxalate (a mixture of 4% uranyl acetate (Polysciences) and 0.15 M oxalic acid (Sigma-Aldrich)) and embedded in a mixture of methylcellulose (Sigma-Aldrich) and uranyl acetate (Polysciences). Samples were observed with a JEM 1400 transmission electron microscope (JEOL Ltd. Tokyo, Japan) at 80 kV, and digital images were acquired with a numeric camera (Morada TEM CCD camera, Olympus, Germany).

EV suspension was deposited on Formvar-carbon-coated 200 mesh copper grids (Agar Scientific, Stansted, UK). The method described by Thery et al. 2018 [7 (link)] was followed for transmission electron microscopy (TEM) analysis. Before contrasted in uranyl oxalate (mixture of 4% uranyl acetate (Polysciences, Warrington, PA, USA) and 0.15 M oxalic acid (Sigma-Aldrich, Schnelldorf, Germany)) and embedded in a mixture of methylcellulose (Sigma-Aldrich, Schnelldorf, Germany) and uranyl acetate (Polysciences, Warrington, PA, USA), EVs were fixed on grids in 2% paraformaldehyde (Sigma-Aldrich, Schnelldorf, Germany) and 1% glutaraldehyde (Polysciences, Warrington, PA, USA). Samples were observed with a JEM 1400 transmission electron microscope (JEOL Ltd. Tokyo, Japan) at 80 kV, and digital images were acquired with a numeric camera (Morada TEM CCD camera, Olympus, Germany).