Morada tem ccd camera

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).

After SEC, 100 µL of the concentrated EVs were mixed with 100 µL of 4% paraformaldehyde (Polyscience, Warrington, PA, USA) to fix the EVs. On the day of analysis, 20 µL of an EVs droplet was covered by a 200-mesh formvar and carbon coated grid (Electron Microscopy Sciences). The EVs droplet was incubated for 20 min at room temperature. After 20 min, the grid was tapped on filter paper to remove excess PBS. Later the grid was rinsed once by gently touching the surface of the grid onto 50 µL of ddH2O droplet and tapped on filter paper to remove the excess water. For negative staining, the grid was transferred onto a droplet of 4% of uranyl acetate (21447 25, Polyscience, Warrington, PA, USA) for 5 min incubation at room temperature. 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).

Suspension of EVs was deposited on formvar-carbon-coated 200 mesh cooper grids (Agar Scientific, Essex, UK) for TEM analysis according to the method described by Thery et al. 200,615. Briefly, EVs on grids were fixed in 2% paraformaldehyde (P6148, Sigma-Aldrich, Schnelldorf, Germany) and 1% glutaraldehyde (O 1909–10, Polysciences, Warrington, USA), before being contrasted in uranyl oxalate [mixture of 4% uranyl acetate (21447–25, Polysciences, Warrington, USA) and 0,15 M oxalic acid (75,688, Sigma-Aldrich, Schnelldorf, Germany)] and embedded in a mixture of methylcellulose (M6385, Sigma-Aldrich, Schnelldorf, Germany) and uranyl acetate (21447–25, Polysciences, Warrington, 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).

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).

EVs isolated using SEC (FF EVs and JAr EVs) were concentrated and deposited on Formvar-carbon-coated 200 mesh copper grids (Agar Scientific, Stansted, UK). The method described by Thery et al., 2018 [35 (link)], was used for transmission electron microscopy (TEM) analysis. In brief, EVs were fixed on grids in 2% paraformaldehyde (Sigma-Aldrich, Schnelldorf, Germany) and 1% glutaraldehyde (Polysciences, Warrington, PA, USA) before being contrasted in uranyl oxalate (a 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). 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).