Oil Red O Staining of Drosophila Midguts

Oil Red O staining was performed as described previously^{32 (link)}. In brief, Drosophila midguts were dissected in 1× PBS and fixed in 4% formaldehyde for 30 min. Midguts were washed three times in 1× PBS, double-distilled water and a 60% isopropanol solution. From the stock solution of Oil Red O (Sigma-Aldrich; 0.1% solution in isopropanol), a working solution was prepared by mixing 6 ml of 0.1% Oil Red O in isopropanol and 4 ml of double-distilled water. Midguts were incubated for 20 min in this solution and then washed in 60% isopropanol and water. The midguts were mounted in Vectashield mounting medium with DAPI (Vector Laboratories) and were imaged by confocal microscopy. Images were captured with the Zeiss LSM 510 confocal system and processed with LSM Image Browser and Adobe Photoshop.