Starvation-Induced Autophagy Visualization

For the starvation experiments, young third instar larvae were washed twice in deionized water and placed for 4 h either on a regular diet medium (fed condition) or in a filtered solution of 20% sucrose in PBS to induce autophagy (starved condition) [56 (link)]. Antibody and phalloidin staining were performed as described previously [57 (link)]. The samples were imaged with a 63x magnification (oil immersion) using a Leica TCS-SP2 confocal microscope and the LCS software. The primary antibodies used in this study were the following: rabbit polyclonal against D. melanogaster Ref(2)P protein [58 (link)], and rabbit monoclonal anti-Cathepsin L (ab133641, Abcam). The appropriate Cy3-conjugated secondary antibodies were purchased from Jackson Immunoresearch Laboratories.
Lysotracker-Red staining on fat bodies was performed as in ref. [56 (link)]. Images were obtained with a fluorescence microscope (Nikon Eclipse 90i) controlled by Nikon Software (Universal Imaging Corp.) using a 60x Plan-Neofluor oil objective.