Lipid Accumulation Assay in Worms

To investigate lipid accumulation in worms, we performed staining using a previously established method with slight modifications [22 (link)]. We examined lipid accumulation in worms of various experimental ages, including four-, five-, six-, and seven-day-old worms subjected to different feeding strategies (OP50, OP50 + LL100933, OP50 + LL12007, LL100933, and LL12007 feeding). Briefly, worms fed different feeds were washed thrice with M9 buffer. The specimens were immobilized in 50% isopropanol solution in phosphate-buffered saline (PBS) for 15 min on ice. The Oil Red O stock solution (0.5 g per 100 mL in isopropanol; Sigma-Aldrich, St. Louis, MO, USA) was diluted with distilled water (dH₂O) to create a 60% working solution, which was then filtered through a 0.2 μm membrane filter. Immobile worms were placed in a functional solution and incubated at 25 °C for 20 min. After staining, immobilized worms were rinsed with M9 buffer containing 0.5% Triton X-100 and placed on glass slides for imaging. Worms were randomly selected for observation [23 (link)]. Imaging was performed using a BX53 microscope fitted with a DP73 color camera (Olympus, Tokyo, Japan). The dye intensity was then measured using the ImageJ software (v1.53), as described previously [24 (link)].