7800 120 kv electron microscope

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.

FBS-sEV and XFS-sEV preparations were fixed by adding 2% PFA in 0.1 mol/L phosphate buffer (pH 7.4) in the same volume of EV resuspension buffer. Five μl drops of EVs were then adsorbed for 10 min on formwar-carbon-coated copper grids. Subsequently, grids were rinsed in PBS and negatively stained with 2% uranyl acetate for 5 min at room temperature. Stained grids were finally embedded in 2.5% methylcellulose for improving preservation and air-dried before examination. Electron micrographs were acquired using a Hitachi 7800 120 kV electron microscope (Hitachi, Tokyo, Japan) working at 100 kV, equipped with a Megaview G3 digital camera and Radius software (EMSIS).

A549 cells were washed out twice in 0.1 M cacodylate buffer and fixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde (Electron Microscopy Science, Hatfield, PA, USA), for 1 h at room temperature. The cells were postfixed in 1% osmium tetroxide for 1 h and 1% aqueous uranyl acetate for 1 h. Subsequently, samples were dehydrated through a graded ethanol series and embedded in resin (Poly-Bed; Polysciences, Inc., Warrington, PA, USA) for 24 h at 60 °C. Ultrathin sections (60 nm) were cut and counterstained with 5% uranyl acetate in 50% ethanol. Electron micrographs were acquired at Hitachi 7800 120Kv electron microscope (Hitachi, Tokyo, Japan) equipped with a Megaview G3 digital camera and Radius software (EMSIS, Munich, Germany). For ultrastructural analysis, 30 randomly taken micrographs for each treatment were examined for mitochondria morphology. The results represent the mean of two independent experiments.