Precision deltavision elite microscope

Fluorescence images of parasites were captured using a GE Applied Precision Deltavision Elite microscope with 100x 1.4NA objective and with a sCMOS camera and deconvolved with the SoftWorx software. Chromatic calibration of the microscope was performed prior to imaging experiments. For immunofluorescence assays, parasites were fixed on slides using 4% paraformaldehyde (ProSciTech) (127 (link)) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). After blocking in 3% bovine serum albumin (Sigma Aldrich) the cells were incubated for 1 h with rabbit polyclonal anti-PfERD2 (1:2000) (128 (link)), mouse monoclonal anti-HA (Cedarlane, CLH104AP, 1:2000), goat anti-human Hb (1:1000, Cedarlane) or rabbit polyclonal anti-BiP (1:1000) (125 (link)). Bound antibodies were then visualized with either Alexa Fluor-594 anti-rabbit, anti-mouse or anti-goat IgG and Alexa Fluor-488 anti-mouse or anti-rabbit IgG diluted 1:1000 (Cedarlane). Parasites were mounted in Vectashield (Vecta Laboratories) containing 0.1 μg/ml 4', 6–diamidino-2-phenylindole (Dapi, Invitrogen). Images shown represent a single optical slice from a deconvolved z-stack. For the LysoTracker labeling experiment, PfPX1-GFP parasites were incubated for 2 h with 75 nM of LysoTracker Red DND-99 (Invitrogen).

Fluorescence images of parasites were captured using a GE Applied Precision Deltavision Elite microscope with 100×1.4NA objective and with a sCMOS camera and deconvolved with the SoftWorx software. Pearson’s correlation coefficients were calculated with the Fiji software^{49 (link)} on regions of interests of image stacks, including zero-zero pixels and without thresholding. For the colocalisation analysis between PfSAC1 and the ER marker Bip, Mander’s correlation coefficients were calculated with the Fiji software on regions of interests of image stacks, with manual thresholding. Chromatic calibration of the microscope was performed prior to imaging experiments. For immunofluorescence assays, parasites were fixed on slides using 4% paraformaldehyde (ProSciTech)^{50 (link)}. After blocking in 3% bovine serum albumin (Sigma Aldrich) the cells were incubated for 1 hour with rabbit polyclonal anti-ERD2 1:2000^{34 (link)}, mouse monoclonal anti-HA 1:2000 (Cedarlane, HA.C5), or rabbit polyclonal anti-Bip 1:500 (Sabrina Absalon and Jeffrey Dvorin, unpublished). Bound antibodies were then visualised with Alexa Fluor-594 anti-rabbit IgG and Alexa Fluor-488 anti-mouse IgG diluted 1:1000. Parasites were mounted in Vectashield (Vecta Laboratories) containing with 0.1 μg/ml 4′, 6–diamidino-2-phenylindole (Dapi, Invitrogen). Images shown represent the maximum projection of all z stacks.