Ultrastructural Identification and Colocalization of Interacting Proteins in the Murine Cochlea by Post-Embedding Immunogold Transmission Electron Microscopy.
Verification of the presence and location of a protein within tissue can be accomplished by western blotting and immunohistochemistry, using either paraffin or frozen sections. Affinity purification by reciprocal coimmunoprecipitations using the tissue of interest can demonstrate the existence of an interacting pair of proteins. Ultimately, the ability to visualize the interaction at the cellular level is desired. Precise location(s) of interacting proteins in situ can be accomplished by ultrastructural localization with high-quality primary antibodies and small-particle-size Au-conjugated secondary antibodies. Visualization can be obtained with a transmission electron microscope fitted with a high-resolution camera permitting magnifications that exceed 2 × 10(5), and, to date, resolution capability of 20+ Mpixels, thus enabling localization of the target protein to within nanometers of the actual location. Here, we report the method by which immunolocalization at the level of the electron microscope is accomplished using the post-embedding technique, i.e., performing antibody labeling of proteins on ultrathin sections of tissue embedded in acrylic resin.
Department of Otolaryngology-HNS, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL, 33612, USA.
Margaret C
MC
Harvey
Department of Otolaryngology-HNS, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL, 33612, USA. bsokolow@health.usf.edu.
Bernd H A
BH
Sokolowski
D000818:Animals; D003051:Cochlea / Q000378:metabolism / Q000648:ultrastructure*; D006046:Gold; D007150:Immunohistochemistry; D051379:Mice; D046529:Microscopy, Electron, Transmission; D025941:Protein Interaction Mapping / Q000379:methods*; D016610:Tissue Embedding; D016707:Tissue Fixation
D006046:Gold
Cochlea; High-resolution imagery; Immunogold; Interacting proteins; Post-embedding technique; Transmission electron microscopy