Fat body staining for glycogen (PAS) and triglycerides (Nile Red)

Third instar larval fat bodies were dissected in ice cold 1 × PBS (10 × PBS:1.37 M NaCl, 27 mM KCl, 100 mM Na₂HPO_4, 18 mM KH₂PO₄, pH 7.4), and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature (RT). For Periodic acid Schiff (PAS) staining tissue was washed in 1% BSA in PBS, incubated in periodic acid solution (Sigma-3951, 1 g/dl) for 5 min at room temperature, washed with 1% BSA in PBS incubated in Schiff’s reagent (Sigma-3952) for 15 min at room temperature and again washed with 1% BSA in PBS. For Nile Red staining tissue was washed in PBS, incubated in PBS containing 0.2 µg/ml Nile Red (Sigma-072485) and 0.5 µg/ml Hoescht 33342 (Sigma-94403) in PBS. Stained tissue was mounted in 80% glycerol (PAS staining) or in 80% plus 5% propyl gallate (Sigma-P3130). Images of PAS-stained fat bodies were taken using bright-field microscopy. Images of Nile-Red-stained fat bodies were obtained on a Leica TCS SC5 Laser Scanning Confocal Microscope. Confocal images are projections of multiple sections obtained using FIJI is Just Image J. The FIJI Analyze Particles function was used to determine the total area of lipid droplets stained by Nile Red, and % area was determined by dividing the total area of lipid droplets by the total area of the fat body.