Improved whole-mount immunofluorescence protocol for consistent and robust labeling of adult Drosophila melanogaster adipose tissue

Energy storage and endocrine functions of the Drosophila fat body make it an excellent model for elucidating mechanisms that underlie physiological and pathophysiological organismal metabolism. Combined with Drosophilas robust genetic and immunofluorescence microscopy toolkits, studies of Drosophila fat body function are ripe for cell biological analysis. Unlike the larval fat body, which is easily removed as a single, cohesive sheet of tissue, isolating intact adult fat body proves to be more challenging, thus hindering consistent immunofluorescence labeling even within a single piece of adipose tissue. Here, we describe an improved approach to handling Drosophila abdomens that ensures full access of the adult fat body to solutions generally used in immunofluorescence labeling protocols. In addition, we assess the quality of fluorescence reporter expression and antibody immunoreactivity in response to variations in fixative type, fixation incubation time, and detergent used for cellular permeabilization. Overall, we provide several recommendations for steps in a whole mount staining protocol that results in consistent and robust immunofluorescence labeling of the adult Drosophila fat body. SUMMARY STATEMENTOptimization of adult Drosophila fat body fluorescence microscopy via modifications of tissue handling, fixation, and permeabilization steps in a whole mount immunolabeling protocol.