Dm5000b compound microscope

Worms were examined on a Leica DM5000B compound microscope. Images were taken with a Retiga 2000R camera and analyzed using ImageJ software. Individual images were stitched together using the MosaicJ plug-in [79] (link).

Whole sporophyte images were obtained using a Nikon Eclipse 80i compound light microscope equipped with a DS-Ri1 digital camera.
Thick sections (1–1.5 µm) of sporophytes processed for transmission electron microscopy (TEM) and in resin blocks were mounted on glass slides and stained with 1.5% toluidine blue in distilled water to monitor the spore stage and integrity using light microscopy. Digital images were captured on a Leica DM5000 B compound microscope.

For experiments using total male scent, a synchronized population of EG4883 hermaphrodites [29 (link)] was raised on small population plates at 20°C until they were 48 hours post L1 arrest. Worms were transferred to either control plates or plates conditioned with four young males. For heat stress experiments, small population plates were shifted to 29°C for 24 hours.
For sperm guidance experiments with single ascarosides and cocktails of two ascarosides, the ascarosides were diluted in water and hermaphrodites were singled onto prepared plates in the same manner as heat stress and recovery experiments. These were single-blind experiments. In all sperm guidance experiments, worms were immobilized with sodium azide and mounted on 2% agarose pads. Images were taken with a Retiga 2000R camera mounted on a Leica DM5000B compound microscope and analyzed using ImageJ software. Individual images were stitched together using the MosaicJ plug-in [48 (link)]. Analyze Particles was used to count fluorescent sperm. An automatic thresholding program (MaxEntropy) [49 ] was used to determine image thresholds.

Most of the phenotypes reported here can be counted under a dissecting microscope. When closer examination was necessary, we used a Leica DM5000B compound microscope fitted with a Retiga 2000R camera. To stitch together individual images, we used the MosaicJ plugin [106 (link)] with ImageJ software. To view adult worms under the compound scope, we mounted them on agarose pads by picking individuals into a drop of 100 mM sodium azide.

Cla-1(ola285) was isolated from a visual forward genetic screen designed to identify mutants with abnormal localization of ATG-9::GFP in the interneuron AIY. OlaIs34 animals, expressing ATG-9::GFP and mCherry::RAB-3 in AIY interneurons, were mutagenized with ethyl methanesulfonate (EMS) as described previously [102 (link)], and their F2 progenies were visually screened under A Leica DM 5000 B compound microscope with an oil objective of HCX PL APO 63×/1.40–0.60 for abnormal ATG-9::GFP localization. SNP mapping [32 (link)] was used to map the lesion corresponding to the ola285 allele to a 6.6- to 11.3-Mbp region on chromosome IV. WGS was performed at the Yale Center for Genome Analysis (YCGA) and analyzed on www.usegalaxy.org using “Cloudmap Unmapped Mutant workflow (w/ subtraction of other strains)” as described [33 (link),34 (link)]. The ola285 allele was sequenced by using Sanger sequencing (Genewiz), and the genetic lesion confirmed as a single T-to-A nucleotide substitution in Exon 15 of cla-1L that results in a missense mutation I5753N. Complementation tests were performed by generating ola285/cla-1(ok560) trans-heterozygotes. The ola285 allele failed to complement cla-1(ok560).

A census of oocytes in the gonad and embryos in the uterus was performed using an established protocol^{58 (link)}. Approximately 30 synchronized L1 larvae prepared by alkaline hypochlorite treatment were pipetted onto either control NGM plates or NGM plates prepared with 2 nM medip#1, as described above. Every 2 h, beginning at 48 h release from post L1 arrest, 25 worms from each condition were examined on a Leica DM5000B compound microscope. The number of oocytes that completely spanned the gonad in both the anterior and posterior arms, as well as the number of fertilized embryos in the uterus, were counted. In addition, the fraction of worms that had ovulated at least once was noted.