22] Subcellular localization of Rab17 by cryo-immunogold electron microscopy in epithelial cells grown on polycarbonate filters

Abstract

This chapter discusses the subcellular localization of Rab17 by cryo-immunogold electron microscopy in epithelial cells grown on polycarbonate filters. High-resolution cryo-immunogold EM is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells because aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Fixed material is embedded in gelatin, cryosectioned, mounted on Formvar-coated grids, and labeled. A typical labeling protocol is described based on the work by Slot and Geuze during which the sectioned material is exposed first to antibody, then to protein A-gold particles. If the primary antibody does not bind to protein A, incubation with a secondary antibody that does bind protein A can be added. The precise reaction conditions used for immunogold labeling may vary depending on the antibody and are determined by trial and error.