Transmission electron microscopy (TEM) sample preparation and imaging

Cells grown on Thermanox coverslips (Nunc) were prepared as described⁶⁶ and adapted for cell monolayers. Cells were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde (Electron Microscopy Sciences EMS, 16400 and 15700) in 0.1 M Hepes buffer pH 7.4 for 1 h at room temperature and then overnight at 4 °C. After washes in cold 0.15 M cacodylate buffer pH 7.4 (Sigma Aldrich, CO250) the cells were post-fixed in 1.5% potassium ferrocyanide (Sigma Aldrich, 60280) and 2% osmium tetroxide (EMS, 19110) in 0.15 M cacodylate buffer pH 7.4 on ice for 30 min. The cells were then washed with double-distilled water (ddH2O) and immersed in filtered 1% thiocarbohydrazide (TCH, EMS, 21900) solution for 10 min. After this step, the cells were stained in 2% osmium tetroxide for 30 min, rinsed again in ddH₂O, and stained with 1% uranyl acetate at 4 °C overnight. After washes with ddH₂O, cells were stained with Walton’s lead aspartate (Sigma A9256 and EMS 17900) solution at 60 °C for 30 min and dehydrated in a graded alcohol series. After flat‐embedding in Epoxy resin (SERVA Electrophoresis GmbH 21045) samples were cured at 60 °C for 12 h. Coverslips were detached from the resin using repetitive freeze−thaw in liquid nitrogen. A region of interest containing cells was selected (~1 × 1 mm), cut, and mounted on an aluminum pin. For transmission electron microscopy (TEM), serial thin sections (60 nm) were cut using an ultramicrotome (Ultracut 7, Leica, Vienna) and imaged using a Spirit 120 kV (FEI, now THF, Eindhoven) at magnifications between 1700× and 8200× equipped with a side-mounted camera (Veleta, EMSIS Gmbh).