Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation

Imaging of gap junction proteins, the connexins, has been performed in tiss ue culture cells both by labeling of connexins with immunocytochemical tags and by cloning and expressing chimeras of connexins and fluorescent protei ns such as Green Fluorescent Protein. These two approaches have been used t o gain information about protein localization or trafficking at light micro scopic resolution. Electron microscopy provides higher resolution; however, analysis of electron micrographs of unlabeled connexins has been generally limited to recognition of gap junction structures. Immunolabeling of gap j unction proteins in whole cells at the electron microscopic level has been difficult to achieve because of the fixation sensitivity of most gap juncti on antibodies. To obtain reasonable sensitivity, immunoperoxidase procedure s are typically employed, and these suffer from relatively poor resolution. Here we describe the combination of tyramide signal amplification techniqu es and fluorescence photooxidation for higher resolution immunolocalization studies for correlative light and electron microscopic imaging. By using c orrelative microscopy, we can not only localize connexin pools or structure s, but also discover what other cellular substructures interact with gap ju nction proteins. The use of tyramide signal amplification techniques is nec essary to increase fluorescence levels that have decreased due to increased specimen fixation required to maintain cell ultrastructure. The fluorescen ce photooxidation technique provides a high-resolution method for staining of proteins in cells. Unlike colloidal gold-based methods, fluorescence pho tooxidation allows for three-dimensional localization using high-voltage el ectron microscopy. Microsc. Res. Tech. 52:331-343, 2001. (C) 2001 Wiley-Lis s, Inc.