Abstract
Lysosomes play a central role in signaling, nutrient sensing, response to stress, and the degradation and recycling of cellular content. Defects in lysosomal digestive enzymes or structural components can impair lysosomal function with dire consequences to the cell, such as neurodegeneration. A number of methods exist to assess lysosomal stress in the model Drosophila, such as specific driver and reporter strains, transmission electron microscopy, and the investigation of gene expression. These methods, however, can be time consuming and, in some cases, costly. The procedure described here provides a quick, reliable, and low-cost approach to measure lysosomal stress in the Drosophila brain. Using fluorescence confocal microscopy and the LysoTracker staining, this protocol allows for the direct measurement of lysosome size and number. This method can be used to assess lysosomal stress under a number of different genetic and environmental scenarios in the Drosophila brain.