Lipid droplet staining

For Nile Red staining, adult fat bodies and guts were dissected in PBS, fixed in 4% paraformaldehyde for 20 minutes, washed twice with PBS and mounted in fresh Nile Red solution with DAPI (0.5 mg/ml Nile Red (ThermoFisher, cat N1142) stock solution diluted 1000x with PBS supplemented with 30% glycerol). For LipidTox staining, adult fat bodies were dissected in PBS, fixed in 4% paraformaldehyde for 20 minutes, washed once with PBST and twice with PBS and stained with 50x dilution of HCS LipidToxTM Green (ThermoFisher, cat. H34475) in PBS. After LipidTox staining, fat bodies were washed with PBS, stained with DAPI and mounted in Fluoromount-G (SouthernBiotech, cat. 0100-01). Images were collected using Olympus Fluoview FV3000. Quantification of surface area occupied by lipid droplets was performed using cellSens Dimension Desktop (Olympus).