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.
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.
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