Dp73 high performance camera

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.

FVs released during the schizont rupture of PbWT and PbAAT1KO parasites were isolated from the culture supernatant as described previously (59 (link), 62 (link)). In brief, C57BL/6 mice infected with PbWT or PbAAT1KO parasites were sacrificed when blood parasitemia reached around 15 to 20%. Infected blood was collected at the late trophozoite stage on the previous night around 22:00 h, centrifuged at 1,000 × g for 5 min, and washed twice with RPMI 1640 containing 10% fetal bovine serum (FBS) to remove the plasma and buffy coat. Parasitized RBCs were then resuspended in 10 volumes of RPMI 1640 containing 10% FBS followed by overnight incubation at 37°C in a CO₂ incubator. Giemsa-stained smears were prepared to monitor the maturation of schizonts, and FVs were isolated from the parasite cultures the next morning around 09:00 h. For this, the cultures were centrifuged two to three times at 200 × g for 5 min to remove the RBCs. The supernatant collected was centrifuged at 400 × g for 10 min to pellet the FVs devoid of merozoites. The FV pellets were then washed twice with 1 mL of PBS by centrifuging at 3,000 × g for 3 min. The purity of intact FVs was examined under brightfield using an inverted Olympus IX83 microscope with a DP73 high-performance camera and a 100× lens objective, followed by storage at −20°C.