Combined Cy3 / Nanogold Conjugates for ImmunocytoChemistry and in Situ Hybridization

Abstract Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab’ immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling. We now describe Fab’ and streptavidin probes containing both Nanogold® and the fluorescent cyanine dye, Cy3. F(ab’)2 Goat anti-Mouse IgG and F(ab’)2 goat anti-rabbit IgG fragments were reductively cleaved to Fab’ fragments using dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA), which selectively reduce the F(ab’)2 hinge disulfide bonds, with 5 mm EDTA to prevent reoxidation. Fab’ fragments were isolated by gel filtration (coarse gel: GH25, Amicon) then labeled with Monomaleimido- Nanogold® which reacts site-specifically with thiols. Streptavidin was labeled using Mono- Sulfo-NHS-Nanogold® at pH 7.5. Nanogold® conjugates were isolated by gel filtration (Superose-12 column, Pharmacia), then reacted with excess Cy3 monofunctional NHS ester (labeling kit, Amersham Life Sciences) at pH 7.5; dual-labeled conjugates were isolated by gel filtration (Superose-12).