Immunofluorescence Analysis of Malaria Parasites

Immunofluorescence analysis for asexual stages, gametocytes, ookinetes, and sporozoites was carried out by fixing the cells with 4% paraformaldehyde and 0.0075% glutaraldehyde, followed by permeabilization with 0.1% Triton X-100 and subsequent treatment with 0.1 M glycine. Blocking was performed with PBS containing 2% BSA for 3 h. The incubation for primary antibodies was carried out in the same blocking buffer for 6 h, followed by the addition of secondary antibodies for 3 h^{77 (link)}. For the oocyst, the mosquito gut was fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X-100, followed by blocking in 4% BSA^{78 (link)}. For exo-erythrocytic stages, HC-04 cells infected with sporozoites were fixed with 4% paraformaldehyde, permeabilized with 0.01% Triton X-100 and blocked with PBS containing 1% BSA^{79 (link)}. Parasite GS-specific polyclonal sera were used at 1:250 dilution. Anti-UIS4 antibody (Origene, AB0042-200) was used at 1:1000 dilution. FITC-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific, A24501) was used at 1:250 dilution. Rabbit anti-goat AF594 (Thermo Fisher Scientific, A-11080) was used at 1:400 dilution. Images were captured with 20x/60x/100x objectives using Olympus IX83 microscope with DP73 high-performance camera.