Plasmodium knowlesi A1-H.1 parasites were fixed with a mixture of 4% paraformaldehyde and 0.075% glutaraldehyde and used for further immunostaining procedures. The fixed parasites were permeabilized with 0.1% Triton X-100 in PBS for 30 min at RT. The slides were then co-stained with mouse serum against either PvMTRAP or PkMTRAP as well as each rabbit serum against P. knowlesi merozoite surface protein 1–19 (PkMSP1–19) (Lee et al., 2023 (link)), P. knowlesi Duffy binding protein (PkDBP) (Hart et al., 2023 (link)), P. vivax rhoptry neck protein (PvRON2) (Arévalo-Pinzón et al., 2011 (link)), and P. vivax rhoptry-associated membrane antigen proteins (PvRAMA) (Lu et al., 2014 (link)) as subcellular localization makers for merozoite surface, microneme, rhoptry neck, and rhoptry body, respectively. After washing, the IgG binding was probed with Alexa Fluor 568-conjugated anti-mouse IgG (H+L) and Alexa Flour 488-conjugated anti-rabbit IgG (H+L) antibody (1:500, Invitrogen), whereas nuclei were stained with 4′,6-diaminidino-2-phenylindole (DAPI) (1:1000, Invitrogen). The slides were mounted with ProLong Gold antifade reagent (Invitrogen) and visualized under oil immersion using a Nikon C2 confocal microscope (Nikon Instrument Inc., City, NY, USA) equipped with a 60× oil objective. The acquired images were prepared using ImageJ.
Free full text: Click here
