Ab133641

Antibody and phalloidin stainings were performed as described previously [25 (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 [54 (link)], mouse monoclonal against Flag tag (Clone M2, Sigma-Aldrich) and rabbit monoclonal anti-Cathepsin L (ab133641, Abcam). The appropriate Cy3-conjugated secondary antibodies were purchased from Jackson Immunoresearch Laboratories.
Lysotracker staining on tissue was performed as in ref. [55 (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.
Image analysis and processing were done with Fiji/ImageJ (National Institute of Health) and Photoshop CS6 (Adobe).For experiments carried out in HeLa cells, the following antibodies were used: anti-β-tubulin monoclonal antibody (Sigma-Aldrich), anti-p62 monoclonal antibody (H00008878-M01, Novus Biologicals), anti-UBPY. Dimethyl sulfoxyde (DMSO) and Hoechst #33342 were from Sigma-Aldrich and Bafilomycin A1 (#tlrl-baf1) was purchased from Invivogen.

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.