Digital camera

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/).

Segments of VN obtained from three MD patients were fixed in phosphate-buffered 2% glutaraldehyde solution for 12 h at 4°C. Specimens were then post-fixed in 1% osmium tetroxide for 3 h, washed with phosphate buffer solution, dehydrated in increasing concentrations of ethanol, immersed in 100% propylene oxide, and embedded in EPON resin blocks (Shell Chemical, USA). Ultrathin sections (70 nm) were made from the blocks using a diamond knife on an ultramicrotome (EM UC7; Leica) and stained with uranyl acetate and lead citrate. Transverse tissue sections were viewed and imaged using a transmission electron microscope at 80 kV accelerating voltage (Tecnai G2 Spirit; FEI, USA) equipped with a digital camera (Gatan, USA).