Use of Anti-fluorophore Antibody to Achieve High-Sensitivity Immunolocalizations of Transporters and Ion Channels

Richard A. Coleman ^1*, Jie Liu ¹ and James B. Wade ¹

¹ Physiology Department, School of Medicine, University of Maryland-Baltimore, Baltimore, Maryland

^* To whom correspondence should be addressed. E-mail: rcoleman{at}umaryland.edu.

Submitted on January 19, 2006
Accepted on 28 February 2006

	Abstract

We have discovered that the immunoreactivity of the fluorophore, Alexa Fluor 488 (Molecular Probes invitrogen detection technologies, Carlsbad, CA), survives glutaraldehyde and osmium tetroxide fixation, and epoxy resin embedding and etching. We have developed new localization methods that, for the first time, take advantage of this property. The antigen is localized in cryosections using suitable primary antibody and an Alexa Fluor 488-conjugated secondary antibody. Cryosection fluorescence can be photographed for later correlation with EM findings, then the sections are further fixed with glutaraldehyde and OsO₄, if desired, and flat-embedded in epoxy resin. Semi-thin sections are etched completely with sodium ethoxide, while thin sections are partially etched, then Alexa Fluor 488 is localized with rabbit anti-Alexa Fluor 488 (Molecular Probes) and goat anti-rabbit conjugated to Alexa Fluor 488 (LM), or to colloidal gold (EM). A second antigen may also be localized using Alexa Fluor 568. When used without postfixation, these methods produce high resolution semi-thin, or even thin sections that retain a high level of fluorescence for LM observations. These methods allow highly sensitive immunolocalizations in tissue while preserving cell fine structure through traditional fixation and epoxy embedding. In demonstration of the methods, we describe the localization of the thiazide-sensitive sodium/chloride cotransporter (NCC) and the epithelial sodium channel (-ENaC) in rat kidney.

Key Words: immunogold, localization, NCC, ENaC, rat, kidney, light microscopy, electron microscopy, Alexa Fluor 488

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