Nile Red staining of neutral lipids and imaging

Fat body and gut tissues were dissected, fixed in 4% Para-formaldehyde in PBS for 30 min at room temperature. Fixed tissues were then rinsed twice in PBS, incubated for 30 min in 1:1000 dilution of 0.05% Nile Red prepared in 1 mg/ml of Methanol, and finally mounted in Antifade mountant with DAPI. To quantify LD size, the area of the 10 largest LDs per fat body cell was measured using ImageJ. This was repeated in at least three independent samples for each fly strain. Images were acquired with Zeiss LSM 510 confocal microscope and processed using Adobe Photoshop CS6.