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Combined Cy3 / Nanogold Conjugates for ImmunocytoChemistry and in Situ Hybridization

Published online by Cambridge University Press:  02 July 2020

Richard D. Powell ,
Vishwas N. Joshi ,
Carol M. R. Halsey ,
James F. Hainfeld ,
Gerhard W. Hacker ,
Cornelia Hauser-Kronberger ,
Wolfgang H. Muss  and
Peter M. Takvorian
Richard D. Powell
Affiliation:
Nanoprobes, Incorporated, Stony Brook, NY11790.
Vishwas N. Joshi
Affiliation:
Nanoprobes, Incorporated, Stony Brook, NY11790.
Carol M. R. Halsey
Affiliation:
Nanoprobes, Incorporated, Stony Brook, NY11790.
James F. Hainfeld
Affiliation:
Biology Department, Brookhaven National Laboratory, Upton, NY11973
Gerhard W. Hacker
Affiliation:
Medical Research Coordination Center, University of Salzburg, Austria
Cornelia Hauser-Kronberger
Affiliation:
Institute of Pathological Anatomy, Salzburg Federal Hospital, Salzburg, Austria.
Wolfgang H. Muss
Affiliation:
Institute of Pathological Anatomy, Salzburg Federal Hospital, Salzburg, Austria.
Peter M. Takvorian
Affiliation:
Biological Sciences Department, Rutgers University, Newark, NJ07102.
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Extract

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).

Type
Biological Labeling and Correlative Microscopy
Information
Microscopy and Microanalysis , Volume 5 , Issue S2: Proceedings: Microscopy & Microanalysis '99, Microscopy Society of America 57th Annual Meeting, Microbeam Analysis Society 33rd Annual Meeting, Portland, Oregon August 1-5, 1999 , August 1999 , pp. 478 - 479
Copyright
Copyright © Microscopy Society of America

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References

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6. This work was partly supported by NIH SBIR grant 4R44 GM56090.Google Scholar