Cell Biology Techniques for Studying Drosophila Peripheral Glial Cells

Glial cells are essential for the proper development and functioning of the peripheral nervous system (PNS). The ability to study the biology of glial cells is therefore critical for our ability to understand PNS biology and address PNS maladies. The genetic and proteomic pathways underlying vertebrate peripheral glial biology are understandably complex, with many layers of redundancy making it sometimes difficult to study certain facets of PNS biology. Fortunately, many aspects of vertebrate peripheral glial biology are conserved with those of the fruit fly, Drosophila melanogaster With simple and powerful genetic tools and fast generation times, Drosophila presents an accessible and versatile model for studying the biology of peripheral glia. We introduce here three techniques for studying the cell biology of peripheral glia of Drosophila third-instar larvae. With fine dissection tools and common laboratory reagents, third-instar larvae can be dissected, with extraneous tissues removed, revealing the central nervous system (CNS) and PNS to be processed using a standard immunolabeling protocol. To improve the resolution of peripheral nerves in the z-plane, we describe a cryosectioning method to achieve 10- to 20-µm thick coronal sections of whole larvae, which can then be immunolabeled using a modified version of standard immunolabeling techniques. Finally, we describe a proximity ligation assay (PLA) for detecting close proximity between two proteins-thus inferring protein interaction-in vivo in third-instar larvae. These methods, further described in our associated protocols, can be used to improve our understanding of Drosophila peripheral glia biology, and thus our understanding of PNS biology.