Colocalization of PfPFD-6 and MSP-1 in Pf3D7

Thin blood smears of mixed stage Pf3D7 cultures at 5% parasitemia were fixed in methanol for 45 min at −20 °C, permeabilized with 0.05% PBS/Tween 20, and blocked with 5% (w/v) BSA in PBS. For colocalization studies, mouse anti‐PfPFD‐6 (1 : 250) and rabbit anti‐MSP‐1 (1 : 250) [52 (link)] were added as primary antibodies and incubated for 2 h at room temperature. Alexa Fluor 488‐conjugated anti‐rabbit (1 : 500, red color; Molecular Probes, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 546‐conjugated anti‐mouse (1 : 500, green color; Molecular Probes) were used as secondary antibodies. The parasite nuclei were counterstained with DAPI (40, 60‐diamidino‐2‐phenylindole; Invitrogen) and mounted with a coverslip. The slides were examined using a confocal microscope (Olympus, Shinjuku, Tokyo, Japan) with a 9 100 oil‐immersion objective.