Computational protocol: APOLs with low pH dependence can kill all African trypanosomes

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Protocol publication

[…] Samples were incubated in fixative (2% paraformaldehyde (PFA, Applichem), 2.5% gluteraldehyde (GA, EMS) in 0.15M Sodium Cacodylate (Sigma-Aldrich) buffer (pH7.4) at room temperature (RT) for 30 min. Fixative was removed by washing 5 x 3 min in 0.15M cacodylate buffer and samples were incubated in 1% osmium (OsO4, EMS), 1.5% potassium ferrocyanide (Sigma-Aldrich) in 0.15M cacodylate buffer for 40 min at RT. This was immediately followed by a second incubation in OsO4 (1% Osmium in double distilled H2O (ddH2O) for 40 min at RT. After washing in ddH2O for 5 x 3 min, samples were incubated overnight at 4°C in 1% Uranyl Acetate (UA, EMS). Uranyl acetate was removed by washing in ddH2O for 5 x 3 min and subsequently dehydrated and embedded as indicated above. Embedded samples were then mounted on aluminium SEM stubs (diameter 12 mm) and coated with ~8nm of Platinum (Quorum Q150T ES). FIB-SEM imaging was performed using a Zeiss Auriga Crossbeam system with Atlas3D software. The Focused Ion Beam (FIB) was set to remove 5 nm-sections by propelling Gallium ions at the surface. Imaging was done at 1.5 kV using an ESB (back-scattered electron) detector. 3D reconstruction and segmentation were generated from the images stacks using Fiji ImageJ (NIH, USA) and ilastik softwares. […]

Pipeline specifications

Software tools ImageJ, ilastik
Applications cryo-EM, Microscopic phenotype analysis
Organisms Trypanosoma brucei, Homo sapiens, Trypanosoma cruzi, Papio papio, Mus musculus
Diseases Infection, Drug-Related Side Effects and Adverse Reactions