Protocol to analyze 3D neurodegenerative vacuoles in Drosophila melanogaster

Vacuole formation is a key hallmark of age-dependent neurodegeneration in the Drosophila brain. Here, we present a protocol to analyze 3D neurodegenerative vacuoles in the whole-mount Drosophila melanogaster brain. We describe steps for whole-brain dissection, staining, 3D imaging, and z-stack image processing using Fiji ImageJ. We then detail procedures for annotating and 3D-reconstructing neurodegenerative vacuoles with WEBKNOSSOS and Python, and performing statistical analysis in Python. This protocol enables measurement of parameters such as the number and volume of each vacuole. For complete details on the use and execution of this protocol, please refer to Elguero et al.¹.