Imagexpress automated imaging system nano imager

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.

Proteasome inhibition was performed using bortezomib (Selleckchem, catalog number S1013) as previously described^{18 (link),22 (link)}. Synchronized L1 animals were plated on 10 cm (for RNA extraction) or 6 cm NGM plates (for phenotypic analyses and transgene expression measurements), and grown for 44 h or 48 h at 20 °C depending on the assay. 10 mM stock solution of bortezomib in DMSO was added to reach a final concentration of 20 µM per plate. The same volume of DMSO was added to the control plates. Plates were dried and worms incubated for 30 min, 4, 21, or 25 h at 20 °C. Imaging was performed using Zeiss AxioImager M1 compound microscope or ImageXpress automated imaging system Nano imager (Molecular Devices, LLC), and analyzed using FIJI program. For RNA extraction, animals were washed off the plates using M9, washed with M9 and collected in TRI reagent (Molecular Research Center, Inc.). ZIP-1::GFP expression was analyzed and imaged on a Zeiss LSM700 confocal microscope run by ZEN2010 software.

Two thousands synchronized L1 worms were mixed with 6 μl fluorescent beads (Fluoresbrite Polychromatic Red Microspheres, Polysciences Inc.), 25 μl 10X concentrated OP50 E. coli, 500,000 N. parisii spores and M9 (total volume 300 ul). This mixture was then plated on 6 cm NGM plates, allowed to dry for 5 min and then incubated at 25 °C. After 5 min, plates were shifted to ice, washed with ice-cold PBST and fixed in 4% paraformaldehyde. Animals were imaged using ImageXpress automated imaging system Nano imager (Molecular Devices, LLC). Fluorescence was analyzed in FIJI program.