Gm. Hand et al., Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation, MICROSC RES, 52(3), 2001, pp. 331-343
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.