A solid-phase immunostaining protocol for high-resolution imaging of delicate structures in the Drosophila larval central nervous system (CNS).

INTRODUCTIONThis protocol describes a method for mounting and immunostaining Drosophila larval tissue in preparation for high-resolution fluorescent imaging of fine structures in the central nervous system (CNS). Affixing the tissue directly to the coverslip and then moving the coverslip between wash solutions provides a simple solid-phase method of immunostaining that assists in preserving fine structures. This method also easily allows for manipulations and/or viewing of the live sample prior to fixation if desired. Finally, putting the tissue in direct contact with the coverslip places fine structures immediately adjacent to the objective lens. We also briefly describe a method to create three-dimensional (3D) models of confocal Z-stacks in order to better characterize fine structures by measuring their volume and obtaining 3D Cartesian coordinates in space.