In Vitro Liver-Stage Malaria Parasite Assay

To study liver-stage development, in vitro cultured human hepatoma (Huh7) cells34 (link) were infected with 10,000 sporozoites using standard techniques as described previously25 (link). After 24, 48 or 70 h, liver-stage parasites were fixed and permeabilized with ice-cold methanol and stained with the DNA-dye Hoechst 33342 (Invitrogen), rabbit anti-UIS4 antibodies (1:500 dilution35 (link)), and mouse anti-HSP70 antibodies (1:300 dilution36 (link)). Primary antibodies were detected by fluorescently labelled goat anti-mouse/rabbit IgG Alexa Fluor 488/546 conjugated antibodies (1:1,000 dilution, Invitrogen). Liver-stage parasites were counted and images recorded on a Zeiss AxioObserver Z1 epifluorescence microscope, equipped with a Zeiss AxioCam MRm camera, and processed minimally with Fiji31 (link).