Ds qi1mc

Sudan Black B staining of lipids was performed on animals fed ad libitum (AL) or starved (BD) for 48 h. Worms were grown at 25°C from the L1 to the L4 stage and then shifted at 20°C. About 400 worms per sample were washed 4 times in M9 buffer and once with PBS + 20% Tween. The supernatant was removed, and 200 μL of PBS was added to the BD samples and 200 μL PBS plus 20 μL of 0.5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) was added to the AL samples. Fresh 1% paraformaldehyde in PBS was added to the worm pellet and incubated for 30 min at room temperature with stirring. Worms were then subjected to three cycles of freeze-thawing in liquid nitrogen and a 37°C water bath, incubated for 10 min on ice, and washed with M9. AL and BD worms were pooled, washed sequentially with 25%, 50%, and 70% ethanol, and then incubated overnight in Sudan Black B solution (50% saturated solution; 15 mg/mL in 70% ethanol). Animals were washed again in 70%, 50%, and 25% ethanol, resuspended in glycerol, and mounted animals on slides. Worms were imaged and photographed using a Nikon Eclipse 80i (Zeiss) microscope equipped with a monochrome digital camera (DS-Qi1Mc).