Em fc6 cryo ultramicrotome

Samples were prepared according to the Tokuyasu method (Tokuyasu, 1973 (link)). Briefly, C. desiccata cells were cultured for 24h in N-deprived medium and fixed in 2% glutaraldehyde and 0.1% acroleine in growth medium for 1h. Following that, cells were washed in growth medium and embedded in 10% gelatin in water. The gelatin was hardened at 4 °C, post-fixed overnight with the fixation medium described above, washed in cacodylate buffer and cut into 0.5mm pieces. The embedded cell pieces were then incubated overnight in 2.3M sucrose, frozen in liquid nitrogen, and cut into 80–90nm slices with an EM FC6 cryo-ultramicrotome (Leica Microsystems). After 30min incubation in blocking solution (0.5% gelatin, 0.5% BSA, 0.2% glycine, 0.1% Tween-20 in PBS), slices were incubated for 2h with anti-ACS2 polyclonal rabbit antibodies in blocking solution (1:150 dilution). Slices were washed with PBS containing 0.2% glycine, incubated for 30min in 10nm colloidal-gold-conjugated goat anti-rabbit antibodies (Electron Microscopy Sciences), diluted 1:20 in blocking solution, and washed in PBS and bi-distilled water. Labelled sections were stained with 2% uranyl acetate, embedded in methyl cellulose, and observed in a Tecnai Spirit Transmission Electron Microscope (FEI) operating at 120kV. Images were recorded using an Eagle 2K×2K CCD camera (FE).

Vitreous sectioning was performed as described before [34 (link)]. SPRF tubes containing the vitrified sample were fixed in a pre-cooled (−145°C) EM FC6 cryo-ultramicrotome (Leica Microsystems, Vienna). The sample was trimmed to a rectangular shaped block of 70–100 μm base and app. 50 μm height using a 20° cryo trimming diamond knife (CT1303; Diatome, Biel, Switzerland). A 25° cryo diamond knife (MT9876; Diatome, Biel, Switzerland) with a clearance angle of 6° was used to get ribbons of cryo-sections at a nominal cutting feed of 50 nm and at cutting speeds of 10 mm/s. Ribbons of cryo-sections were attached to pre-cooled 600 mesh EM grids with an eyelash using electrostatic charging with the Crion™ (Leica Microsystems, Vienna) [41 (link)].

Immuno-electron microscopy was performed by cryosectioning and immunolabeling^{40 (link)}. In brief, HeLa cells transiently expressing GFP-TECPR1 were fixed in two steps, first with 8% paraformaldehyde (PFA) and 0.4% glutaraldehyde (GA) in PHEM buffer for 5 min at room temperature and second with 4% PFA/0.2% GA in PHEM buffer for 30 min at room temperature. Cells were embedded in 2% gelatin, infused with 2.3 M sucrose at 4 °C, mounted onto pins and plunge-frozen in liquid nitrogen. Ultrathin cryosections were cut using a Leica EM FC6 cryo-ultramicrotome. Sections were immunolabeled with anti-GFP (1:50) and protein A conjugated to 10 nm gold. Samples were viewed on a Philips CM120 BioTwin Transmission Electron Microscope.