Fei tecnai g2 spirit twin

Electron microscopy was performed at the Facility for Electron Microscopy Research (FEMR) at McGill University. 10 µL of protein samples were deposited and adsorbed for 1 min onto carbon-coated grids that were glow discharged before the application of proteins. Grids were glow discharged for 1–2 min in a vacuum evaporator (Edwards Vacuum Carbon Coater E306) before adding the protein samples to improve quality of sample analysis. Assembled NFL as well as SacsJ were diluted to 0.24 µg/µL and processed for imaging by TEM. Excess protein was removed by wicking the edge of the grid on a piece of Whatman paper. The samples were then negatively stained for 1 min using 10 µL of 2% (w/v) uranyl acetate and examined with a Cryo-TEM (FEI Tecnai G2 Spirit Twin) (Thermo Fisher Scientific, St Laurent, QC, Canada) using an accelerated voltage of 120 KV. Images were acquired with a CCD camera (Gatan Ultrascan 4000 4 k × 4 k CCD Camera System Model 895). The diameters of the assembled filaments were measured on enlarged TEM micrographs using ImageJ software (National Institute of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/ accessed on 1 May 2020).

GMECs were washed with PBS and collected by centrifugation, then fixed by 2.5% glutaraldehyde at room temperature for 1 h followed by further fixation at 4 ℃ overnight. After post-fixation by 1% osmium tetroxide for 1 h, cells were dehydrated with graded ethanol solutions (30, 50, 70, 80, 95, and 100%) and sodium sulfate anhydrous treated acetone. After infiltrating with a 1:1 mixture of acetone and EPON resin for 1.5 h, 100% EPON resin overnight, cells were then embedded in epoxy resin and cured at 60 ℃ for 48 h. Ultra-thin sections of 70 nm were cut by a Leica ultramicrotome. The characteristic changes of ferroptosis were observed under TEM (FEI Tecnai G2 Spirit Twin, ThermoFisher Scientific, USA) after double staining with uranyl acetate and lead nitrate.