420 transmission electron microscope

Cells were fixed in a freshly-prepared solution containing 3% formaldehyde (prepared from paraformaldehyde) and 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min at room temperature (RT). Cells were then harvested using a scraper, collected into a tube and centrifuged at 800 g for 5 min at RT. The supernatant was aspirated, while cells were resuspended in 4% gelatin warmed aquatic solution followed by a spin down at 800 g for 5 min at RT and cooled on ice. Under a stereoscope the solidified cell pellet with gelatin was extracted, cut into small fragments (1–2 mm³) and transferred into 0.1 M phosphate buffer, pH 7.4 at 4 °C. The cell-gelatin fragments were then dehydrated in graded series of ethyl alcohol, followed by propylene oxide (PO) treatment, infiltrated gradually in a mixture of Epon/Araldite resins diluted in PO and finally embedded in fresh epoxy resin mixture. Ultrathin epoxy sections (70-90 nm thickness) were cut on a Leica Ultracut R ultramicrotome, equipped with a Diatome diamond knife, and mounted onto 200-mesh copper grids. Ultrathin sections were observed with a Philips 420 transmission electron microscope and micrographs were taken with an Olympus Megaview G2 CCD camera.

At 1 month post-injection, haSyn PFF-treated WT and KO-aSyn mice (n = 4/genotype) were perfused transcardially with 0.1M PBS (pH 7.2) at 37°C and then with 4% paraformaldehyde/1% glutaraldehyde. The brain was removed and the ipsilateral striatum was processed for EM analysis as described in Supp. Methods (Online Resource 16). In all EM procedures, the grids were examined in a Philips 420 transmission electron microscope at an acceleration voltage of 60 kV and photographed with a Megaview G2 CCD camera (Olympus SIS, Münster, Germany) and iTEM image capture software. In order to assess myelin integrity in the PFF-injected mice striatum, we quantified the g-ratio (ratio of inner axonal diameter to total outer diameter) and the % of axons with decompacted myelin [43 (link)]. For these quantifications, at least 100 randomly selected myelin sheaths that were cross-sectioned completely without artifacts and could be classified without doubt were counted. Semi-automated analysis of randomly selected myelin sheaths was carried out using a plug-in for ImageJ software as previously described [7 (link)].

Negatively stained electron micrographs were obtained by adding 5 μl of EVs in PBS solution, onto Formvar-coated 400 mesh copper grids, fixing with 0.5% glutaraldehyde (5 μl), and staining with a 2% aqueous uranyl acetate solution (Sigma-Aldrich, USA) for 2 min. Grids with stained EVs were examined in a Philips 420 transmission electron microscope at an acceleration voltage of 60 kV and images were acquired with a Megaview G2 CCD camera (Olympus SIS, Munster, Germany).