Alexafluor 546 conjugated anti mouse

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.

Cells grown on coverslips were fixed in 3.7% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 15 min, blocked with 10% FBS in PBS for 2 h, and incubated with primary antibodies overnight. For the detection of autophagosomes or lysosomes, antibodies against LC3 or Lamp1, respectively, were used. Cells were washed and incubated with Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 633-conjugated anti-mouse, Alexa Fluor 488-conjugated anti-mouse, Alexa Fluor 405-conjugated anti-mouse, Alexa Fluor 546-conjugated anti-rabbit, or Alexa Fluor 546-conjugated anti-mouse secondary antibodies (all from Thermo Fisher) for 2 h and visualized under a confocal microscope (LSM 510, Carl Zeiss, Thornwood, NY, USA). Numbers of puncta were counted using ImageJ analysis software (NIH, Bethesda, MA, USA).

Cells were seeded in 4-well dishes (35/10 mm; Greiner Bio-One North America, Inc.) and cultured in respective conditions. After 18 h culturing, cells were treated with 0.5% DMSO (control) or 15.5, 31, or 46.6 µM CX-4945 for indicated incubation time. Subsequently, cells were washed twice with PBS, fixed with 3.7% paraformaldehyde solution (PFA) for 20 min, washed twice with PBS, and incubated for 30 min at the room temperature with permeabilization and blocking solution (5% BSA, 0.1% Triton X 100, and 0.5% Tween 20 in PBS). Subsequently, the solution was discarded, and the cells were washed three times with PBST (0.1% Tween 20 in PBS) and incubated with the primary antibodies (anti-DHFR or anti-TS, both 1:100) in PBST for 24 h at 4°C. After washing with PBST (0.5 ml/compartment), the cells were protected from light and incubated with Alexa Fluor 546-conjugated anti-mouse diluted 1:500 (ThermoFisher Scientific, United States) for 1 h at the room temperature. After washing, the cells were incubated in 1 µl/ml Hoechst 33342 in PBST for 15 min. The dye solution was discarded, and the cells were washed with PBST (0.5 ml/compartment). Pictures were taken using the Olympus Fluoview FV1000 confocal laser scanning microscope (CLSM, Olympus, Center Valley, PA, United States).