N. parisii spores were prepared as previously described [33 (link)]. 0.5 million spores were mixed with OP50-1, M9, and 1,200 synchronized L1 animals, and the mix was top-plated onto 6-cm NGM plates; at least two replicates (two plates) per genotype were set up per infection assay. Three independent infection assays were set up for each timepoint described below. Animals were infected at 25°C for 3 h or 30 h before fixation in either 4% paraformaldehyde or 100% acetone. Fixed worms were incubated at 46°C overnight with FISH probes conjugated to the red Cal Fluor 610 fluorophore that hybridize to N. parisii ribosomal RNA (Biosearch Technologies, Hoddeson, United Kingdom). 3 hpi samples were analyzed for N. parisii sporoplasms using a AxioImager M1 compound microscope (Zeiss). 30 hpi samples were analyzed for N. parisii meronts using a COPAS Biosort machine (Union Biometrica, Holliston, MA) where FISH signal for each worm was normalized to the length of the worm using time-of-flight measurements.
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