Cm100 biotwin transmission electron microscope

Co-cultures were fixed in 2% (wt/vol.) glutaraldehyde (Polysciences, Eppelheim, Germany) for 24 h. Samples were post-fixed using osmium tetroxide (Sigma-Aldrich, St. Louis, MO, USA)/potassium ferrocyanide (Sigma-Aldrich) for 30 min. Next, samples were embedded in Epon 812 (SERVA, Heidelberg, Germany) and polymerised at 37°C for 16 h followed by 56°C for 24 h. Thick sections (0.5 μm) were stained with toluidine blue (Sigma-Aldrich). Ultrathin sections (60 nm) were stained with uranyl-acetate (Sigma-Aldrich) in methanol and lead citrate (Sigma-Aldrich). Imaging was performed using a CM100 Biotwin transmission electron microscope (FEI, Eindhoven, the Netherlands).

Cell samples for Transmission Electron Microscopy (TEM) were collected at 2 h, 7 h and 24 h p.i., and fixed with 0.2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h. The fixed cells were rinsed twice for 5 min in 0.1 M cacodylate buffer at room temperature followed by post-fixation in 1% osmium tetroxide, 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate at 4°C for 30 min. The cells were then washed with Milli-Q water, dehydrated through serial incubation in a graded ethanol series (30%, 50%, 70% and 100%) and lastly embedded in EPON resin and polymerized at 37°C for 16 h followed by 58°C for 24 h. Ultrathin sections (80 nm) were cut with an UC7 ultramicrotome (Leica, Vienna, Austria) and contrasted using 5% uranyl acetate for 20 min, followed by Reynolds lead citrate for 2 min. Images were recorded with a CM100 Biotwin transmission electron microscope (FEI, Eindhoven, The Netherlands) operated at 80 kV using a Morada digital camera. Image processing was performed with FIJI (https://fiji.sc/).