Tecnai 12 biotwin electron microscope

Briefly, control and GRASP55 knockout cells were grown in 35 mm plastic dishes to 80% confluence. Cells were then fixed with 1% Glutaraldehyde (Electron Microscopy Sciences) in 0.2 M HEPES pH 7.3 at RT for 1 h. The fixative was replaced with 1% BSA in PBS, and cells were carefully detached using a plastic cell scraper, collected into microfuge tubes, and centrifuged to obtain the pellet. All samples were then washed three times in 0.2 M HEPES pH 7.3 and post‐fixed 30 min in 1% Osmium Tetroxide in the dark at 4°C in the same buffer. They were then washed three times in distilled water and post‐fixed 25 min in 1% Osmium Tetroxide and 1.5% Potassium Ferrocyanide in the dark at room temperature in HEPES 0.2 M pH 7.3. After washing three times in distilled water, they were stained with 0.5% uranyl acetate over night at 4°C. The pellets were dehydrated in graded steps of ethanol (50, 70, 90, and 100%), 2 times with 100% of acetone, and embedded into Epon. Thin sections (60 nm) were cut on a Leica UC7 ultramicrotome and examined with 120 kV Philips Tecnai 12 Biotwin electron microscope (FEI, Eindhoven, The Netherlands) using a VELETA digital camera.

The cells were fixed using 2.5% glutaraldehyde in 0.1 M phosphate buffer, osmicated in 1% osmium tetroxide, and dehydrated in increasing ethanol concentrations. During dehydration, 1% uranyl acetate was added to the 70% ethanol to enhance ultrastructural membrane contrast. After dehydration the cells were embedded in Durcupan. 70 nm ultrathin sections were cut on a Leica ultramicrotome, collected on Formvar coated single-slot grids, and analyzed with a Tecnai 12 Biotwin electron microscope (FEI).