Transgenic flies expressing GFP-mCherry-Atg8a (obtained from the Bloomington  Drosophila  Stock Center)  under the control of the  neuronal nSyb-GAL4 driver are used for assaying autophagic flux

Brains from 10-day-old animals are dissected in ice-cold 1X PBS

The dissected brain are transferred to a glass slide

The remaining 1X PBS is removed from the slide

A drop of DAPI Fluoromount is added and the brain mounted with a coverslip

The dissected brain is imaged immediately within 5 minutes using a Zeiss confocal microscope under a 63X objective lens, and a line scan performed

For quantification, the intensity of GFP and mCherry is measured per puncta using ImageJ, and the ratio between GFP and mCherry calculated