Stage-synchronized worms were harvested with M9 buffer with polyethylene glycol 4000 (PEG 4000) (0.01%; Tokyo Chemical Industry, Japan) and washed three times with M9 buffer. Worms were fixed with 4% paraformaldehyde (158127, Sigma-Aldrich, MO, USA) solution in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4; AM9624, Thermo Fisher Scientific, MA, USA) for 45 min with gentle agitation. After fixation, worms were washed with PBS containing 0.01% PEG 4000 twice with gentle agitation for 5 min for each washing step. Fixed worms were stored in 70% ethanol at 4°C overnight. Worms were placed on a 2% agarose pad on a slide glass, and the worms were soaked in a drop of 4′,6-diamidino-2-phenylindole (DAPI) solution (2 ng/μl) (62248, Thermo Fisher Scientific, MA, USA) in VECTASHIELD antifade mounting media (Vector Laboratories, CA, USA). After covering the agar pad with a coverslip, worms were incubated for at least 30 min in the dark before imaging.

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