Oso4 in h2o

PfVPS45-2xFKBP-GFP parasites 8 h after induction of the knock sideways (with 250 nM rapalog) and the control cells grown without rapalog were Percoll-enriched and fixed with 2.5 % glutaraldehyde (Electron Microscopy Sciences) in 50 mM cacodylate buffer pH 7.4 for 1 h at room temperature. For some experiments the cells were first treated with 1 HU Tetanolysin in Dulbecco DPBS (Pan Biotech) for 10 min at 37 C to release the host cell cytosol as described (Mesen-Ramirez et al., 2016) and washed 3 times in DPBS prior to fixing. Cells were post fixed with 2 % OsO4 in H2O (Electron Microscopy Sciences) for 40 minutes at 4 C in the dark, contrasted with 0.5 % uranyl acetate (Agar Scientific) for 30 minutes at room temperature and dehydrated through increasing concentrations of ethanol. Following embedding in epoxy resin (EPON) (Carl Roth GmbH & Co. KG) samples were cut into 60 nm sections with an Ultracut UC7 (Leica) and examined with a Tecnai Spirit transmission electron microscope (FEI), equipped with a LaB6 filament and operated at an acceleration voltage of 80 kV. The EM image .emi/.ser-files were converted to 8 bit TIFF files using the TIA Reader Plugin for imageJ. Batch conversion was performed with simultaneous contrast enhancement using a saturation value of 5.