Uranyl acetate

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).

TEM analyses were performed exactly as described recently [44 (link)]. Briefly, 7.5 µL of 1:100 diluted VLP preparations were mixed with 1 µL aqueous contrasting solution, containing 1% methyl cellulose (w/v; Sigma Aldrich) and 2% uranyl acetate (w/v; Polysciences, Warrington, FL, USA). Following incubation for 10 min, 0.5 µL droplets were placed on 200 mesh copper grids (Plano, Wetzlar, Germany) and dried at room temperature, allowing included VLPs to adhere to the films’ surfaces. Images were taken on a JEM 1400 Plus electron microscope (JEOL, Tokyo, Japan), equipped with a 4096 × 4096 pixel CMOS camera (TemCam-F416; TVIPS, Gauting, Germany), and run at an operating voltage of 120 kV. Image acquisition software EMMENU (Version 4.09.83) was used for taking 16-bit images. Image post-processing was performed with the software ImageJ (Version 1.52b; National Institutes of Health, Bethesda, MD, USA).

To identify the distribution of the mitochondria in the muscle fibres, biceps and extensor carpi radialis muscle were removed cut in pieces of 1 mm³ and immerse fixed in 4% PFA and 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 (4°C, 48 hr). Tissue blocks were contrasted using 0.5% OsO₄ (Roth, Germany; RT, 1.5 hr) and 1% uranyl acetate (Polysciences, Germany) in 70% ethanol (RT, 1 hr). After dehydration tissue blocks were embedded in epoxy resin (Durcopan, Roth, Germany) and ultrathin sections of 40 nm thickness were cut using a Leica UC6 ultramicrotome (Leica, Wetzlar, Germany). Sections were imaged using a Zeiss 906 TEM (Zeiss, Oberkochen, Germany) and analysed using ITEM software (Olympus, Germany).

N. benthamiana leaf tissues with or without RSV infection (14 dpi) were prepared, subjected to vacuum infiltration, and fixed immediately with 2.5% glutaraldehyde (Sigma, G5882) in 0.1 M PBS overnight at 4 °C. Samples were washed three times with PBS, post-fixed with 1% OsO₄ (Sigma, O5500), rinsed three times with PBS again, dehydrated in a graded ethanol series followed by the replacement of ethanol with acetone, and embedded in SPI-PON812 resin (SPI Science, 90,529–77–4). The ultrathin sections were stained with 2% (w/v) uranyl acetate (Polysciences, 21,447–25) and 2.6% (w/v) lead citrate (Sigma, 15,326). Images were observed and captured using a transmission electron microscope (TEM; Hitachi H-7650, Japan) at 80 kV (Guan et al. 2022 ).

Serial sectioning, electron microscopy and tomography, and image processing were performed as described previously (Doroquez et al., 2014 (link)). Briefly, serial plastic sections (70-nm thick) were collected on slot grids covered with Formvar support film, post-stained with saturated solution of uranyl acetate (0379, Polysciences, Inc., Warrington, PA) for 15 min, and Reynold’s lead citrate (Lead nitrate - 17900, EMS, and Sodium citrate - S-279, Fisher) for 7 min, and imaged using a Tecnai F20 (200 keV) or F30 (300 keV) transmission electron microscope (FEI, Hillsboro, OR) equipped with a 2K ×2K charged-coupled device (CCD) camera. For large overviews of sections, we acquired montages of overlapping high-magnification images. For electron tomography, BSA-coated, 10 nm colloidal gold fiducials (Au - Sigma-Aldrich, St. Louis, MO; BSA - SC-2323, Santa Cruz Biotechnology, Inc.) (Iancu et al., 2006 (link)) were applied to the sections, before acquiring dual-axis tilt series with a tilt range of ±60° with 1° increments around each axis. Automated montage and tilt series acquisition was facilitated by the microscope control software SerialEM (Mastronarde, 2005 (link)). Image processing, such as blending montages and reconstructing tomograms, was performed using various tools from the IMOD software package (Kremer et al., 1996 (link)).

Electron microscopy was performed on adult mice perfused with 4% PFA. The brain was dissected immediately after perfusion and stored in 2% PFA + 2% Glutaraldehyde. A piece of cerebral cortex was cut from a frontal slice of the brain and prepared for EM. Samples were washed in 0.1 M cacodylate buffer (Sigma-Aldrich), incubated for 2 h in 1% osmium tetroxide (Science Services, Munich, Germany), dehydrated in an ascending series of ethanol, and embedded in Epon 812 (Serva). Ultrathin sections were counterstained with uranyl acetate (Polyscience, Eppelheim, Germany) and lead citrate (Riedel-de Haën, Seelze, Germany), and analyzed with a LEO 912 AB OMEGA electron microscope (Leo Elektronenmikroskopie, Oberkochen, Germany).