BODIPY staining and imaging

The abdominal dorsal cuticle was dissected in relaxing buffer (1X PBS, 5 mM MgCl₂ and 5 mM EGTA) using micro-scissors with the adipose tissue attached to the cuticle as described before (Rajan et al., 2017). The samples were fixed with 4% paraformaldehyde in relaxing buffer for 20 min. Subsequently the samples were washed with PBS, permeabilized with 0.1% PBT (PBS + 0.1% TritonX100) in PBS for 30 min. PBT was removed by washing with PBS, three times, 5 min each, before adding BODIPY. For BODIPY staining, 500 μl of BODIPY in PBS (1/500 dilution of a 1 mg/ml stock in DMSO) was added to the samples and the samples were placed on a rotator for 30 min at room temperature. The samples were then washed in PBS, two times, 10 min each, incubated in PBS with DAPI for 10 min, washed two more times in PBS, 10 min each, and then mounted with Vectashield mounting media (VectaShield 1000). Samples were mounted with a bridge using two pieces of scotch tapes (3M) with the adipose tissue facing the cover slip. Samples were imaged using a Zeiss LSM780 confocal microscope. Images were acquired at room temperature.