Multimodal Imaging of Parasite Organelles

For mitochondrial staining, the parasite culture was treated with 75 nM MitoTracker Red CMX-Ros (Invitrogen) for 30 min in fresh media prior to fixation. All cultures were fixed in 4% paraformaldehyde–0.0075% glutaraldehyde on polylysine-treated slides for 30 min. Cells were permeabilized for 10 min in 1% Triton X-100, remaining aldehydes were reduced with 0.1 mg/ml NaBH₄ for 10 min, and cells were blocked for 2 h in 3% BSA. Parasite cytosol was stained with 1:2,000 mouse anti-aldolase, the apicoplast was stained with 1:1,000 rabbit anti-ACP (38 (link)), and lipoamidase was stained with 1:500 rat anti-HA MAb 3F10 (Roche) overnight. After treatment with primary antibodies, the slides were washed and treated with cognate secondary antibodies. The cognate secondary antibodies were 1:2,000 rabbit α-mouse IgG Alexa Fluor 594 (rabbit anti-mouse IgG conjugated to Alexa Fluor 594 diluted 1:2,000), 1:2,000 goat α-rabbit IgG Alexa Fluor 594, or 1:2,000 goat αRat IgG Alexa Fluor 488 (Invitrogen). The slides were washed and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen). Microscopy images were taken with the Zeiss Axio Imager. The PDM channel and PCC values were derived using Volocity software as previously described (39 (link)), and mean PCC values were obtained by analyzing multiple images from two independent experiments for each genotype.