Large Cluster and Combined Fluorescent and Gold Immunoprobes

Richard D. Powell,* James F. Hainfeld,* Carol M. R. Halsey,* David L. Spector,** Shelley Kaurin,** Jennifer McCann,** Roger Craig,~ Fredric S. Fay,~ and Kathryn E. McNamara~

* Nanoprobes, Incorporated, Stony Brook, NY 11790;
** Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724;
~ University of Massachusetts Medical Center, Biomedical Imaging Group, Worcester, MA 01605.

The fluorescein/Nanogold probe was used to label a pre-mRNA splicing factor in HeLa cell nuclei, as a secondary antibody against a monoclonal primary antibody specific for the SC35 splicing factor.² Specific staining was observed by confocal laser scanning microscopy (Fig. 2(a)), in which measured fluorescence intensities were approximately one-half of those found with a commercially available FITC-labeled secondary IgG antibody, and by transmission electron microscopy with silver enhancement (HQ Silver, Nanoprobes)(Fig. 2(b)).

Combined fluorescein/Nanogold anti-mouse Fab' was also used to label vinculin and pericentrin in smooth muscle cells, as a secondary probe for monoclonal antibodies against these proteins.³ Specific staining was observed by fluorescence microscopy with deconvolution analysis.⁴ Measured fluorescence intensities were 0.4 relative to a commercially available fluorescein-labeled IgG secondary antibody. For vinculin, some specific staining was also observed by electron microscopy with silver enhancement. These results indicate that these reagents provide a unique method for visualizing the same molecules at both the fluorescence and the EM level.

Functionalized platinum and palladium clusters 2 to 3 nm in diameter were prepared by reduction of an eight-fold excess of metal acetate with a mixture of the functionalized 1,10-phenanthroline ligands (4) and (5), followed by air oxidation of the surface metal atoms.⁵ Platinum clusters prepared in this manner are shown in Fig. 5. These large clusters were conjugated to goat anti-mouse Fab' fragments using the procedure described above; conjugates were isolated by gel filtration HPLC (Superose-12, Pharmacia). These conjugates were readily silver enhanced: in immunoblots, they exhibited sensitivities equal to Nanogold conjugates, detecting as little as 10 pg of mouse IgG immobilized on nitrocellulose membrane. These larger labels were clearly visible in the electron microscope at 35,000X magnification in the immunolocalization of SC35 in HeLa cells; Nanogold was not visualized under these conditions.

References

1. Hainfeld, J. F., and Furuya, F. R.; J. Histochem. Cytochem., 40, 177 (1992).

2. Spector, D. L., Fu, X.-D., and Maniatis, T.; EMBO J., 10, 3467 (1991) .

3. Moore, E. D. W., et al ; Nature, 365, 657 (1993).

4. Coggins, L. W., and Fay, F. S.; in "Computer Methods and Programs in Biomedicine;" Vol. 22, p. 69; Elsevier Publishers, New York, NY (1986).

5. (a) Schmid, G.; Morun, B., and Malm, J.-O.; Angew. Chem. Int. Ed. Eng., 28, 778 (1989); (b) Schmid, G., et al; J. Amer. Chem. Soc., 115, 2046 (1993).

6. This work was supported by NIH grants 2R44 GM48328 and 2R44 GM49564.

FIG. 1. (a) Ligands used for combined fluorescent /gold labels; (b) Fluorescein/Nanogold-Fab'.

FIG. 2. (a) Confocal laser scanning micrograph of HeLa cells labeled with anti-SC35 monoclonal primary antibody, detected with fluorescein/Nanogold-labeled goat Fab' anti-mouse antibody fragment (X 400) (b) Electron micrograph after silver enhancement (X 18,000).

FIG. 3. 1,10-phenanthroline ligands for preparation of large platinum/palladium cluster complexes.

FIG. 4. Transmission electron micrograph of large platinum clusters (Original mag. X 75,000).

Thanks to the San Francisco Press for allowing us to reproduce this online.