Immunostaining and Dye Penetration Experiments to Define Core Pleated Septate Junction Proteins in Drosophila...

The purpose of these protocols is to evaluate the organization and barrier function of pleated Septate Junctions (pSJs) in the ectodermally-derived epithelial tissues of Drosophila embryos. pSJ form an occluding barrier on the lateral membrane of ectodermal tissues including the epidermis, salivary glands, trachea, and hindgut that is functionally similar to the tight junction in vertebrate tissues. In Drosophila, 30 proteins have been identified that are required for the formation of pSJs. Core pSJ proteins are initially localized along the length of the lateral membrane but become strongly enriched at the apical lateral region of the junction, just basal to the adherens junction, by stage 16 of embryogenesis. By late stage 15 of embryogenesis, these tissues possess a fully functional occluding junction. This paper will describe immunostaining and dye permeability protocols that can be used to interrogate the organization and function of pSJs in control and mutant embryos. Successful employment of these protocols can be used to demonstrate the occluding function of pSJs, and to test whether an unknown gene encodes a core component of the junction.