Axio imager m1 compound microscope

Embryos were mounted in 70 % glycerol, 30 % PBS and DIC pictures were taken using an AxioCam MRc5 camera mounted on a Zeiss Axio Imager M1 compound microscope. Fluorescent images were taken on a Zeiss LSM 710 confocal microscope. Images were processed using Adobe Photoshop software (Adobe, Inc) and Image J software (Abramoff et al., 2004). In some cases different focal planes were merged to show labeled cells at different medial lateral positions in the spinal cord.

Embryos 24 h and older were deyolked in 70% glycerol/30% sterile water using mounting pins. For lateral and dorsal views of the embryo, whole embryos were mounted in 70% glycerol in coverslip sandwiches (24 × 60 mm coverslips; VWR, 48393-106), with 2–4 coverslips (22 × 22 mm; VWR, 16004-094) on either side of the sample to avoid sample compression. For ventral views of putative taste receptors, the trunk was dissected with a razor blade and the head carefully inverted on to a 24 × 60 mm coverslip and a similar coverslip sandwich made. For lateral views of eyes, they were dissected from forebrain using mounting pins and mounted as for whole embryos, but using only 1–2 coverslips each side of the specimen. Cross-sections were cut by hand using a razor blade mounted in a 12 cm blade holder (World Precision Instruments, Cat. #14134). Differential interference contrast (DIC) pictures were taken using an AxioCam MRc5 camera mounted on a Zeiss Axio Imager M1 compound microscope. A Zeiss LSM 710 confocal microscope was used to image embryos mounted in DABCO (1,4-Diazabicyclo[2.2.2]octane, Sigma, D-2522, 2% w/v solution in 80% sterile glycerol) for fluorescent double-labeling experiments. Images were processed using Adobe Photoshop software (Adobe, Inc) and Image J software (Abràmoff et al., 2004 ).

Before imaging, animals were anesthetized with 100 μM levamisole. Samples were imaged using a Zeiss AxioImager M1 compound microscope equipped with Axio Vision 4.8.2 software (Fig 1A, 1B, 1F, 1G, 1I, 2A, 3A, 6E, S1, S2B and S3A) or a Zeiss LSM700 confocal microscope run by ZEN2010 software (Fig 5).

Orsay virus filtrates were prepared as previously described [29 (link)]. A 1:5 dilution of Orsay virus filtrate was mixed with OP50-1, M9, and 1,000 synchronized L1 animals, and the mix was top-plated onto 6-cm NGM plates; at least two technical replicates (two plates) per genotype were set up per infection assay. Four independent infection assays were performed. Animals were infected at 20°C for 18 h before fixation in 4% paraformaldehyde. Fixed worms were incubated at 46°C overnight with two FISH probes, mixed equally, that were both conjugated to the red Cal Fluor 610 fluorophore and hybridize to either Orsay RNA1 or RNA2 genome segments (Biosearch Technologies). Samples were analyzed for Orsay virus infection using a AxioImager M1 compound microscope (Zeiss). 100 animals per genotype, per infection assay, were scored and the percentage of infected animals in the population was quantified.

Orsay virus isolate was prepared as previously described^{11 (link)}. For pals-5p::GFP expression analysis and FISH staining for infection level quantification, L1 animals were exposed to a mixture of OP50-1 bacteria and Orsay virus for 12 h at 20 °C, whereas animals used for ZIP-1::GFP analysis were infected with a high dose of virus for 9 h at 20 °C. pals-5p::GFP reporter expression was analyzed in animals that were anesthetized with 10 mM levamisole. 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 overnight using FISH probes conjugated to the red Cal Fluor 610 fluorophore, targeting Orsay virus RNA1. GFP imaging and FISH analysis were performed using Zeiss AxioImager M1 compound microscope. For qRT-PCR analyses, synchronized L4 animals were exposed to a mixture of OP50-1 bacteria and Orsay virus for 24 h at 20 °C. RNA isolation and qRT-PCR analysis were performed as described below. ZIP-1::GFP expression was analyzed and imaged on a Zeiss LSM700 confocal microscope run by ZEN2010 software.

Orsay viral preps were prepared as previously described [12 (link)]. Synchronized L1 animals were exposed to a mixture of OP50-1 bacteria and Orsay virus for 18 h at 20 °C (Fig 6A). For infection at the L4 stage (Fig 6B), synchronized animals were grown on rde-1 RNAi plates (1,000 animals per plate, 2 plates per strain) for 72 h (pals-17(*) mutants) or 48 h (all other strains) at 20 °C. rde-1 RNAi increases susceptibility to Orsay virus infection [78 (link),79 (link)]. L4 animals were top-plated with a mixture of Orsay virus, OP50-1 and M9, and incubated at 20°C for 24 h. Following incubation, animals were collected and washed in M9, and subsequently fixed in 4% paraformaldehyde for 30 min. Fixed worms were washed and then stained at 46 °C overnight using FISH probes conjugated to the red Cal Fluor 610 or green FAM fluorophore (Biosearch Technologies), targeting Orsay virus RNA1 and RNA2 [80 (link),81 (link)]. Analyses were performed visually using a Zeiss AxioImager M1 compound microscope; at least 300 animals were scored for the presence of the FISH fluorescent signal per strain per replicate. Due to relatively high variation in infection levels between replicates, the percentage of infected wild-type animals was normalized to one. Data analysis was performed using GraphPad Prism 9 software. Normalized and raw data are shown in S1 Table.