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Journal of Histochemistry and Cytochemistry, Vol. 45, 947-956, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

A Covalent Fluorescent-Gold Immunoprobe: Simultaneous Detection of a Pre-mRNA Splicing Factor by Light and Electron Microscopy

Richard D. Powella, Carol M. R. Halseya, David L. Spectorb, Shelley L. Kaurinb, Jennifer McCannb, and James F. Hainfelda
a Nanoprobes, Incorporated, Stony Brook, New York
b Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Correspondence to: Richard D. Powell, Nanoprobes, Inc., 25 East Loop Road, Suite 124, Stony Brook, NY 11790-3350.

Immunoprobes that combine a fluorescent label with a 1.4-nm gold cluster compound have been prepared by covalent conjugation with polyclonal antibody Fab' fragments. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. We find that incorporation of one or more fluorescein moieties into the coordinated ligands of the 1.4-nm Nanogold gold cluster label yields a stable, dual-function immunolabel in which fluorescence quenching is negligible. In a second synthetic strategy, Nanogold and fluorescein were separately covalently conjugated to Fab' fragments to yield a probe with very similar properties. With the use of Fab' fragments, the entire probe is smaller than a whole IgG molecule, and it exhibited excellent penetration properties. It was used to localize the pre-mRNA splicing factor SC35 in the HeLa cell nucleus by both fluorescence and electron microscopy. (J Histochem Cytochem 45:947-956, 1997)

Key Words: immunogold, immunofluorescence antibody, fluorescence microscopy, electron microscopy, immunoprobe, cluster, nucleus


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