Whole-Larva Cryosectioning and Immunolabeling of Drosophila Larvae

Abstract

Resolution in microscopy—the shortest distance between which objects can be distinguished from each other—is crucial for our ability to view details of biological samples. The theoretical resolution limit of light microscopy is 200 nm in the x,y-plane. Using stacks of x,y images, 3D reconstructions of the z-plane of a specimen can be achieved. However, because of the nature of light diffraction, the resolution of the z-plane reconstitutions is closer to 500–600 nm. Peripheral nerves of the fruit fly Drosophila melanogaster consist of several thin layers of glial cells surrounding the underlying axons. The size of these components can be well under the resolution of z-plane 3D reconstructions, thus making it difficult to determine details of coronal views through these peripheral nerves. Here, we describe a protocol to obtain and immunolabel 10-μm cryosections of whole third-instar larvae of the fruit fly Drosophila melanogaster. Cryosectioning the larvae using this method converts visualization of coronal sections of the peripheral nerve into the x,y-plane and brings the resolution down from 500–600 nm to 200 nm. Theoretically, this protocol can also be used with some modifications to obtain cross sections of other tissues.