Immunofluorescence Imaging of Malaria Parasites

Air dried thin smears of P. berghei ookinete or schizont or sporozoites and oocysts, obtained from midgut of P. berghei infected Anopheles stephensi crushed in PBS, were fixed with 4% EM grade paraformaldehyde (Electron Microscopy Science) for 10 minutes. Permeabilisation, blocking and incubation with primary and appropriate secondary antibodies (S4 Table & S5 Table) was performed as described by [41 (link)] with additional last washes with 70% ethanol and absolute ethanol 1 min each, air-dried and mounted in VectaShield (Vectorlabs) containing DAPI (4', 6-diamidino-2-phenylindole) in glycerol for nuclear staining. Parasites were examined either under Delta Vision Epifluorescence microscope (Applied Precision). 100x objective, images were captured with CoolSNAP HQ camera (Photometrics) and deconvoluted using SoftWoRx software (Applied Precision) or under Axioplane2 (Zeiss) 100x objective, images were taken through HAMAMATSU ORCA_ER camera (HAMAMATSU) and Velocity software 4.1.0 (PerkinElmer). Images were processed using Fiji (NIH) as well as SoftWoRx explorer 1.3. Super-resolution images were captured through Elyra PS.1 super-resolution microscope (Zeiss) under 60x objective with sCMOS PCO camera and images were visualized with ZEN Black software (Zeiss) and processed with ZEN LITE software (Zeiss).