Lysotracker ds red dnd 99

For Lyso-Tracker staining, intact guts were removed from flies that had been maintained on 40μM DHM or control food for 20 days (n=5 flies in each group). Dissections were performed in phosphate buffered saline. Each gut was mounted into a custom-made imaging chamber and stained with 1μM of LysoTracker DS Red DND-99 (Invitrogen, Molecular Probes) for 3 minutes. Each preparation was then washed three times with PBS and mounted in mounting medium (Mounting medium, Vectashield, H1200). Imaging was performed using a fluorescence microscope with ×10 objective and ×10 eyepiece. Numbers of LysoTracker-stained puncta were analyzed by Image Pro.Plus6.0.

The experimental Drosophila were cultured for 20 days, and the guts were extracted and stained with 1 μM LysoTracker DS Red DND-99 (Invitrogen, Molecular Probes) for 3 minutes and then washed 3 times with PBS before mounting and performing microscopy.