Megaview g3 digital camera

EV preparations were resuspended in 20 μL PBS (pH 7.4) and fixed by adding an equal volume of 2% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). EVs were then adsorbed for 20 min to Formvar–carbon-coated copper grids by floating the grids on 5 μL drops on parafilm. Subsequently, grids were rinsed in PBS and negatively stained with 2% uranyl acetate for 5 min at RT [44 (link)]. Stained grids were embedded in 2.5% methylcellulose for improved preservation and air dried before examination. Electron micrographs were taken at Hitachi TEM microscope (HT7800 series, Tokyo, Japan) equipped with Megaview G3 digital camera and Radius software (EMSIS, Muenster, Germany).

Primary neuronal cultures from PS19 mice expressing P301S mutant human tau and cultured NSC-34 cells transfected with vehicle or human TDP-43 expressing plasmid were fixed, 40 h after transfection, with 2.5% glutaraldehyde (Electron Microscopy Science) in 0.1 M cacodylate buffer for 1 h at room temperature, post-fixed in 1% OsO₄ for 1 h, 1% tannic acid for 30 min and en bloc stained with 1% uranyl acetate for another hour. Then samples were dehydrated through a graded ethanol series and flat embedded in epoxy resin (Poly-Bed; Polysciences, Inc.) for 24 h at 60 °C. Ultrathin sectioning (50 nm) was performed with Leica ultramicrotomes (Reichert Ultracut, Leica microsystems). Flat-embedded cells were cut parallel to the substrate and counterstained with 5% uranyl acetate in 50% ethanol. Ultrastructural analysis was performed with a Hitachi 7800 120 kV electron microscope (Hitachi) operating at 100 kV using a Megaview G3 digital camera and Radius software (EMSIS). Electron micrographs were taken using the Multiple Image Alignment (MIA) montage and screenshot tools.