Eclipse ni e imaging system

For IFA, thin smears of parasitized RBC on slides were air dried, fixed in 4% PFA in PBS for 10 to 20 minutes, permeabilized in 0.1% (v/v) Triton X-100 in PBS for 10 minutes, and blocked with 3% bovine serum albumin (BSA) in PBS for at least 30 minutes at 4°C. Slides were then probed with the appropriate dilution of primary antibody in a humidified chamber at RT for 1 hour before being washed 3 times in PBS. Secondary antibody conjugated with Alexa Fluor 488 or 594 was added for 1 hour at the appropriate dilution, followed by 3 washes in PBS. Slides were mounted in ProLong Gold Antifade mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) and viewed on a Nikon Eclipse Ni-E imaging system with a Hamamatsu Orca-flash 4.0 digital camera and a Plan apo λ 100×/1.45 oil immersion objective. Images were captured using Nikon NIS-Elements software, generating Z-stack images of individual parasites, using deconvolution options and exporting the image as a tiff file. Alternatively, images were processed using Fiji software [38 (link)]. Identical exposure conditions were used for each wavelength in treated (rapamycin or IMP-1002) and control (DMSO) samples.

For IFA, slides were air dried, fixed in 4 % paraformaldehyde (PFA) in PBS for 10–20 min, permeabilized in 0.1 % Triton X-100 in PBS for 10 min, and blocked with 3 % BSA in PBS for at least 30 min at 4°C. GAP45 detection was achieved using a primary rabbit anti-GAP45-antibody (Rees-Channer et al., 2006 (link)). Slides were mounted in ProLong® Gold Antifade mounting medium containing DAPI (4’,6-diamidino-2-phenylindole), and viewed on a Nikon Eclipse Ni-E imaging system with a Hamamatsu Orca-flash 4.0 digital camera and a Plan apo λ 100x/1.45 oil immersion objective. Images were captured using Nikon’s NIS-Elements imaging software generating Z-stack images of individual parasites, using deconvolution options and exporting the image as a tiff file, which was further edited using Adobe Photoshop.