Oil red o working solution

To measure fat accumulation, we used oil red O staining, using a slightly modified protocol from that previously used [29 ]. Synchronized L4 worms were cultured on NGM plates with different treatments for 5 d at 20 °C. Worms were then collected and washed in M9 with 0.05% Triton X-100 three times. The worms were centrifuged at 560× g for 1 min and the supernatant was removed, leaving approximately 100 µL. Then, 600 µL of 40% isopropanol was added to the worm pellet and rocked at room temperature for 3 min. The above centrifugation steps were repeated. Subsequently, the worms were stained with 600 μL of Oil Red O working solution (Beyotime, Shanghai, China) for 2 h at room temperature at 30 rpm. After washing, worms were imaged by a stereomicroscope (55 × magnification) outfitted with a color camera (S9i, Leica, Wetzlar, Germany). The color picture was converted to an 8-bit file and the whole worm was carefully selected using the polygon selections tool using ImageJ, and its optical density was then quantified.

Paraffin sections of adipose tissue were made and subjected to antigen retrieval and endogenous peroxidase activity blocking. Anti-Plin5 antibody (PA5-114352, Thermofisher, Waltham, MA, USA) was used as the primary antibody, and then the biotin-labeled secondary antibody (1:3000) was incubated for 1 h. The image was taken with an Olympus microscope.
After the frozen section (10 μm) of adipose tissue was made, the section was warmed up at room temperature and was immersed in distilled water. Then it was soaked in 60% isopropanol for 2 min, and was dyed with 5% oil red O working solution (Beyotime, Shanghai, China) for 5 min. It was toned with 60% isopropanol, and was washed and sealed with glycerin gelatin. The sections were observed under a microscope (Olympus, Tokyo, Japan).