Leo 912ab transmission electron microscope

Cells were fixed in 4% paraformaldehyde, washed, permeabilized for 10 minutes with 0.25% saponin, 0.1% BSA in PBS, and blocked (0.2% BSA, 5% Normal Goat Serum, 50mM NH₄Cl, 0.1% saponin, 20mM phosphate buffer, 150mM NaCl). Coverlips were then incubated with primary antibody (anti-UBC9 BD Biosciences or anti-LC3 Novus Biologicals) for 2 hours, washed (0.1% BSA, 0.1% Saponin in PBS) and incubated for 1 hour with secondary antibodies conjugated with nanogold (Nanoprobes). Samples were then fixed with 1% glutaraldehyde for 1 hour and nanogold was enlarged with a gold enhancement solution (Nanoprobes) according to manufacture instructions, postfixed (1% OsO₄, 1.5% potassium ferrocyanide in 0.1M cacodylate buffer pH 7.4), enbloc stained with 1% uranyl acetate over/night at 4°C, dehydrated with ethanol, embedded in EPON 812 and cured in an oven at 60°C for 48 hours. Ultrathin sections (70-90nm) were cut on an ultramicrotome (Leica FC7, Leica microsystem). Grids were stained with uranyl acetate and Sato’s lead solutions and observed in a Leo 912AB Zeiss Transmission Electron Microscope (Carl Zeiss). Digital micrographs were taken with a 2Kx2K bottom mounted slow-scan Proscan camera (ProScan, Lagerlechfeld, Germany) controlled by the EsivisionPro 3.2 software (Soft Imaging System).

Cells were fixed with 2.5% glutaraldehyde in 100 mM cacodylate buffer pH 7.4 for 1 hour at room temperature. After several washes in cacodylate buffer, cells were postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 100 mM cacodylate buffer pH 7.4 for 1 hour on ice. After a rinse in dH₂O, samples were en bloc stained in 0.5% uranyl acetate overnight and dehydrated in increasing concentrations of ethanol and finally embedded in Epon. Samples were cured at 60°C in an oven for 48 h. Epon blocks were sectioned using a Leica EM UC7 ultramicrotome (Leica Microsystems, UK). Ultrathin sections (70 nm) were contrasted with 2% uranyl acetate and Sato's lead solutions and observed with a LEO 912AB Zeiss Transmission Electron Microscope (Carl Zeiss, Oberkochen, Germany). Digital micrographs were taken with a 2k × 2k bottom-mounted slow-scan ProScan camera (ProScan, Lagerlechfeld, Germany) controlled by the EsivisionPro 3.2 software (Soft Imaging System, Münster, Germany).