COMBINED FLUORESCENT AND GOLD IMMUNOPROBES - REAGENTS AND METHODS FORCORRELATIVE LIGHT AND ELECTRON-MICROSCOPY

Immunoprobes which incorporate both a fluorescent label and a 1.4 nm g old cluster compound were prepared by covalent conjugation to Fab' ant ibody fragments of the Nanogold cluster label followed by a fluorescen t moiety. These new immunoconjugates allow the collection of two compl ementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. By using Fab' fragments, the entire pro be is smaller than a whole IgG molecule. A simple fluorescence assay w as used to investigate the fluorescence properties of the new probes. They were used to localize the pre-mRNA splicing factor SC35 in the He La cell nucleus by both fluorescence and electron microscopy, and also for labeling leukocyte microtubules; labeling was imaged using fluore scence microscopy and, after silver enhancement, by a variety of optic al methods and by electron microscopy. Combined Nanogold and Texas Red , Cy3, Lissamine Rhodamine B, and AMCA probes were also prepared, and in preliminary experiments show similar properties to the combined flu orescein and gold cluster probes. The fluorescent and gold cluster pro bes enable a new degree of correlation between fluorescence and electr on microscopy, and may also be used to check labeling of specimens bef ore processing for electron microscopy. Microsc. Res. Tech. 42:2-12, 1 998. (C) 1998 Wiley-Liss, Inc.