Ags160

TEM images were obtained in a JEOL JEM-1400 TEM with an accelerating voltage of 120 kV, equipped with EMSIS Xarosa digital camera with Radius software. Samples were sonicated for 2 min immediately prior to adsorption to the grid. Then, 10 μL of the sample was applied to a freshly glow discharged carbon film grid (400 mesh, AGS160-Agar Scientific Ltd). Grids were glow discharged in a Quorum GloQube System for 15 s at 20 mA. Then, the sample was left to adsorb and dry for 25 min at a room temperature. The statistical analysis of the images was made using open source software ImageJ.

Transmission electron microscopy (TEM) images were obtained in a JEOL JEM-1400 TEM with an accelerating voltage of 120 kV, equipped with EMSIS Xarosa digital camera with Radius software. Samples were sonicated for 2 min immediately prior to adsorption to the grid. Then, 10 μL of the sample was applied to a freshly glow discharged carbon film grid (400 mesh, AGS160-Agar Scientific Ltd). Grids were glow discharged in a Quorum GloQube System for 15 s at 20 mA. Then, the sample was left to adsorb and dry for 25 min at a room temperature. The statistical analysis of the images was made using open source software ImageJ.

EM was performed as previously described (Di Mattia et al., 2018 (link); Wilhelm et al., 2017 (link); Alpy et al., 2013 (link)). Cells grown on carbon-coated sapphire disks were cryoprotected with DMEM containing 10% FCS and frozen at high pressure (HPM 10 Abra Fluid AG). Samples were then freeze-substituted and embedded in lowicryl HM20. Thick sections (∼250 nm) were collected on carbon-coated copper grids (200 Mesh; AGS160; Agar Scientific).
EM grids were placed on a MatTek glass bottom dish in a drop of water and imaged with a spinning-disk confocal microscope (CSU-X1, Nikon, 100× oil objective, NA 1.4). The position of the imaged cells was determined using the asymmetrical center mark of the grid. Then, samples were imaged with a transmission electron microscope (Philips CM12) coupled to an Orius 1000 CCD camera (Gatan). Images were processed and merged with the open-source software Icy (de Chaumont et al., 2012 (link)) using the eC-CLEM plugin (Paul-Gilloteaux et al., 2017 (link)).