Sporozoite staining

To stain for sporozoite proteins, 5 × 10⁴ freshly dissected sporozoites were incubated in DMEM medium or DMEM containing 10% FCS at 37 °C on glass coverslips. After 30 min of incubation, sporozoites were fixed in 4% paraformaldehyde for 10–20 min at room temperature. Fixed sporozoites were permeabilized using ice-cold methanol for 5 min at −20 °C. After washing, sporozoites were blocked and stained as described above. Coverslips were imaged using Zeiss LSM 710 confocal laser point-scanning fluorescence microscope (using ZEN 2 software, Blue edition), using ×63 or ×100 oil objective. The primary antibodies used were goat αPbUIS4 (1:500 dilution, Sicgen, Cantanhede, Portugal); mouse αPbCSP (1:1000 dilution, clone 3D11, MR4); mouse αPfEXP2 (1:300 dilution, clone 7.7, The European Malaria Reagent Repository, Edinburgh, Scotland, UK); rabbit αPfEXP2 (1:300 dilution, a kind gift from the laboratory of Brendan Crabb⁵³) and rabbit αPbTRAP (1:1000 dilution, a kind gift from the laboratory of Joana Tavares⁶⁹) and mouse αPbRON4 (1:300 dilution, a kind gift from the laboratory of Maryse Lebrun⁷⁰).

For colocalization experiments, the plug-in Coloc 2 from FIJI image analysis software was used.