Jan 29, 2024
Assessment of autophagic flux assay in the adult Drosophila brain
- Mel Feany1,2
- 1Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Mel Feany 2024. Assessment of autophagic flux assay in the adult Drosophila brain. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x661lqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94348
Keywords: ASAPCRN
Abstract
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.
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