594 anti rabbit igg

The subcellular localization of NIF4-Ty in P. falciparum was observed under a fluorescence microscope as previously described (83 (link)). Briefly, the NIF4^iKD parasites were fixed with 4% paraformaldehyde/0.0075% glutaraldehyde in PBS at room temperature for 30 min. Fixed parasites were washed with PBS three times, permeabilized with 0.1% Triton X-100 in PBS for 10 min on ice, rinsed with 0.1 mg/mL of sodium borohydride in PBS, and blocked in 5% skimmed milk. Then, the samples were incubated with mouse anti-Ty1 MAb (1:500), the rabbit anti-EXP2 sera (1: 500) as the PVM marker, or the rabbit anti-Pfs16 sera (1:500) as the gametocyte marker. Secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG or 594 anti-rabbit IgG) (Thermo Fisher) were used at 1:500 dilution. The samples were mounted with ProLong Diamond Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher) and observed by Nikon C2 fluorescence confocal laser scanning microscope. Postprocessing and image analysis were performed using Adobe Photoshop and Image J software. At least 20 images were captured for each colocalization experiment, and Pearson’s correlation coefficients were calculated.

We performed immunofluorescence assays using rabbit anti-γH2AX (Ser139) foci monoclonal antibody (Cell Signaling Technology Inc.) to detect the DNA damage and repair [33 (link)]. Laser confocal microscopy (ZEISS LSM710, Oberkohen, Battenburg, Germany) was used to detect the immunofluorescence using 594-anti-Rabbit IgG (Thermo Fisher Scientific, Shanghai, China) and DAPI fluorescent dye (Beyotime).