Quantifying Nematocida parisii Infection

N. parisii spores were prepared as previously described^{63 (link)}. Spores were mixed with food and L1 synchronized animals (a dose of 8 million spores per plate was used for ZIP-1::GFP expression analyses, a dose of 0.5 million spores per plate was in all other assays). Animals were incubated at 25 °C for 3 h (for sporoplasm counting and ZIP-1::GFP analysis), 24 h (for qRT-PCR analysis of IPR gene expression) or 30 h (for pathogen load analysis). For pals-5p::GFP expression analysis, animals were anesthetized with 10 µM levamisole and imaged using Zeiss AxioImager M1 compound microscope. For FISH analysis, animals were collected and fixed in 4% paraformaldehyde for 15–45 min depending on the assay. Fixed worms were stained at 46 °C for 6 h (for ZIP-1::GFP analysis) or overnight (for pathogen load analyses) using FISH probes conjugated to the red Cal Fluor 610 fluorophore, targeting ribosomal RNA. 3 hpi samples were analyzed using Zeiss AxioImager M1 compound microscope; 30 hpi samples were imaged using ImageXpress automated imaging system Nano imager (Molecular Devices, LLC), and fluorescence levels were analyzed using FIJI program. ZIP-1::GFP expression was analyzed and imaged on a Zeiss LSM700 confocal microscope run by ZEN2010 software.