Low viscosity epoxy resin

BMMCs were fixed in 4% formaldehyde/PBS at RT for 1 hour, and stored at 4°C. After washing, cells were post-fixed with 1% glutaraldehyde in PBS for 1h at RT and then rinsed and pelleted in gelatin 12%. Gelatine pieces containing cells were post-fixed with 1% osmium tetroxide reduced with 1.5% potassium ferrocyanide in PBS for 1h, progressively dehydrated in ethanol, and embedded in low-viscosity epoxy resin (Agar Scientific Ltd). Blocks were cured at 60°C for 24h. 70-nm-thin sections were cut using an EM UC6 ultramicrotome (Leica), mounted on copper grids, and stained with uranyl acetate and lead citrate. Sections were examined with a 120 kV TEM (Tecnai 12, Thermo Fischer Scientific) equipped with a 4K CDD camera (Oneview, Gatan).

Ovisacs, dissected as described above, were transferred into carriers type A (6 mm diameter; 200 µm depth) and covered with the flat surface of carriers type B (LEICA Microsystems, Wien, Austria). Notably, the carriers were coated with 1-hexadecene prior to use, and the inside volume of carrier A was filled up with 10% bovine serum albumin (BSA). The carrier sandwich was inserted into the middle plate of a sample cartridge and high-pressure frozen (HP-frozen) with the freezer HPM100 (LEICA Microsystems, Austria). FS was performed in an automatic freeze substitution unit AFS2 (Leica Microsystems, Austria) equipped with a self-made agitation module described elsewhere (Goldammer et al., 2016 (link)). Carriers containing the HP-frozen samples were placed onto the frozen substitution medium (1% OsO₄ in acetone) in Sarstedt tubes. For Selected area electron diffraction (SAED), ovisacs of the TUZ strain from Turkey were also freeze-substituted in acetone in absence of OsO₄. FS took place under agitation at −85 °C, initially for 3 h, but mostly for 10 h overnight. It was followed by warming up to room temperature and embedding in low viscosity epoxy resin (Agar Scientific, Stansted, UK). The temperature/time course was measured and recorded with a K- type thermocouple and USB data logger EL- USB-TC- LCD (Lascar Electronics, Erie, PA, USA) inside a dummy tube filled with acetone.