Drosophila as a Model to Monitor Multi-organ Autophagy.

Autophagy is a conserved lysosome-mediated pathway essential for cellular homeostasis, development, and stress responses. The fruit fly Drosophila melanogaster serves as a powerful model for autophagy research due to its genetic tractability and conservation of human disease genes. This chapter details fluorescence microscopy-based methods to monitor autophagy in multiple tissues, including the brain, midgut, Malpighian tubules, and ovary, under nutrient-replete and starvation conditions. Autophagosome dynamics are visualized using mCherry-Atg8a, while lysosomes are detected with CathepsinL immunostaining; colocalization of these markers in presence and absence of autophagy inhibitors can be used to measure autophagic flux. Clearance of Ref(2)P/p62 is additionally used as a readout for cargo degradation. Stepwise protocols are provided for tissue dissection, fixation, staining, and imaging, with guidance on data analysis and image processing. These approaches enable reproducible assessment of tissue-specific autophagy in vivo, offering valuable tools to investigate its roles in development, stress adaptation, and aging in Drosophila.