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Updated: May 12, 2002

N A N O P R O B E S     E - N E W S

Vol. 3, No. 5          May 12, 2002


This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.

Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing [email protected].

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Gold-Facilitated In Situ Hybridization: A Brightfield Chromogenic Gene Amplification Assay for HER-2/Neu

Detection with Nanogold®-streptavidin followed by silver or gold enhancement has been established as one of the most sensitive methods available for in situ hybridization. When used with tyramide signal amplification, it can detect single copies of a target gene. Tubbs and co-workers now report the development of an in situ hybridization assay for amplification of the HER-2/neu gene, one of the critical prognostic factors for malignancy in breast cancer. This method uses Nanogold-streptavidin with gold enhancement to generate a dense black signal, which is read in the light microscope.

Unlike fluorescence in situ hybridization (FISH), this method allows simultaneous visualization of the underlying morphology, and the black stain is easily distinguished from other commonly used stains. The staining is permanent, and does not photobleach. Furthermore, by controlling the sizes of the signals, interpretation is simplified, as it now depends upon the overall staining pattern rather than spot counting.

Reference:

Tubbs, R.; Pettay, J.; Skacel, M.; Powell, R.; Stoler, M.; Roche, P., and Hainfeld, J.: Gold-Facilitated in Situ Hybridization: A Bright-Field Autometallographic Alternative to Fluorescence in Situ Hybridization for Detection of HER-2/neu Gene Amplification. Am. J. Pathol., 160, 1589-1595 (2002).

Abstract (American Journal of Pathology):
http://ajp.amjpathol.org/cgi/content/abstract/160/5/1589

More information:

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Nanogold® Labeling of Factor XII and its Role in Endothelial Binding

The versatility of covalent Nanogold® labeling was demonstrated this month by Fahdi and co-workers, who used Nanogold to label Factor XII in order to define its binding site on cultured endothelial cells. Although they do not state which labeling reagent they used, the experimental parallel with FITC labeling, and also the retention of binding to negatively charged surfaces via the positively charged N-terminal region, supports labeling at a cysteine residue with Monomaleimido Nanogold. Fluorescence microscopy was used to demonstrate that Factor XII colocalizes with urokinase plasminogen activator receptor and gC1qR on the cell membrane, and electron microscopy with silver enhancement confirmed that most of the labeled Factor XII localized to the endothelial cell membrane. Inhibition studies with antibodies to these proteins and peptides with sequences corresponding to regions of Factor XII showed that Factor XII interacts with the multiprotein assembly of urokinase plasminogen activator receptor, gC1qR, and cytokeratin 1 on endothelial cell membranes.

Reference:

Mahdi, F.; Madar, Z. S.; Figueroa, C. D., and Schmaier, A. H.: Factor XII interacts with the multiprotein assembly of urokinase plasminogen activator receptor, gC1qR, and cytokeratin 1 on endothelial cell membranes. Blood, 99, 3585-3596 (2002).

Abstract (Blood):
http://www.bloodjournal.org/cgi/content/abstract/99/10/3585

Because linking is site-specific and chemically selective, Nanogold labeling may be used in the same manner as fluorescent labeling for a wide variety of biological molecules, many of which cannot be labeled with conventional colloidal gold. Examples include:

More information:

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Gold and Silver Staining - Techniques in Molecular Morphology

A new reference book on immunogold-silver staining is now available, including three chapters written by Nanoprobes scientists on Nanogold®, FluoroNanogold, and silver and gold autometallography of Nanogold. "Gold and Silver Staining: Techniques in Molecular Morphology" edited by Gerhard W. Hacker and Jiang Gu and published by the CRC Press, Boca Raton, FL, also contains chapters on in situ hybridization, autometallographic tracing of gold, silver, bismuth, mercury and zinc, and Immunogold-silver staining for scanning electron microscopy. It includes many protocols, and discusses the applications to cancer research and other fields.

More information (CRC Press):
http://www.crcpress.com/shopping_cart/products/product_detail.asp?sku=1392&parent_id=&pc=/shopping_cart/search/search.asp!

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NanoVan and Image Analysis Yield Novel Protease Structure

Negative stain electron microscopy with NanoVan, and image analysis of 1,300 particles, were used by Franzetti and colleagues to define the structure of a large, tetrahedral, dodecameric protease complex (TET) from archaea. TET, which has broad aminopeptidase activity and can process peptides of up to 30-35 amino acids in length, has a central cavity accessible through four narrow channels (<17 Å wide) and four wider channels (21 Å wide). This is in contrast to other proteolytic complexes described to date, that are made up by rings or barrels with a single central channel and only two openings.

The authors found NanoVan's pH of close to 8 was better suited to their system than that of uranyl acetate; they also found that NanoVan was more resistant to electron beam damage than uranyl acetate.

Reference:

Franzetti, B.; Schoehn, G.; Hernandez, J. F.; Jaquinod, M.; Ruigrok, R. W.; and Zaccai, G.: Tetrahedral aminopeptidase: a novel large protease complex from archaea. EMBO J., 21, 2132-2138 (2002).

Abstract (EMBO Journal):
http://embojournal.npgjournals.com/cgi/content/abstract/21/9/2132

NanoVan is recommended for use with Nanogold because it is based on vanadium and is therefore lighter than heavy metal based stains such as uranyl acetate or lead citrate, and hence makes visualization easier. It may also be mixed with our tungsten-based negative stain, Nano-W, and its opacity "tuned" to suit your application.

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Getting the Best Results with FluoroNanogold

FluoroNanogold is a unique probe containing both Nanogold® and fluorescein, which may be used for correlative fluorescence and electron microscopy labeling, or for checking labeling by fluorescence before EM processing. Because FluoroNanogold combines two labels, which are frequently used in different concentrations under different conditions, some optimization may be required to obtain the best performance in your experiments. Some suggestions for obtaining the cleanest signal and lowest background:
  • Adjusting concentration: higher concentrations of FluoroNanogold can improve the signal-to-noise ratio. Changes in the concentration of primary antibody may also affect the fluorescence signal; therefore, this should be tried at a lower or higher dilution.

  • Adjusting camera exposure: manual control of exposure can greatly help in reducing apparent background. FluoroNanogold is frequently compared with commercially available fluorescently labeled IgG conjugates, which since they are larger and more highly labeled, give brighter fluorescence. If automatic exposure adjustment is allowed with FluoroNanogold-stained specimens, the greater exposure can lead to higher apparent backgrounds. Fixing camera exposure manually can be used to overcome this effect.

  • Reducing hydrophobic interactions: the following reagents added to the incubation or wash buffers can help reduce hydrophobic interactions of FluoroNanogold with cell or tissue components:
    • Adding a small amount of 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling CO2 into an aqueous suspension of triethylamine with stirring) to the wash buffer after FluoroNanogold incubation (Reference: Safer, D.; Bolinger, L., and Leigh, J. S.; J. Inorg. Biochem., 26, 77 (1986).
    • Adding a small amount (0.1 % to 1 %) of detergent, such as Tween-20, or Triton X-100.
    • Adding a small amount (0.1 % to 0.5 %) of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.
More information:

Original papers:

  • Powell, R. D.; Halsey, C. M. R.; Spector, D. L.; Kaurin, S. L.; McCann, J.;, and Hainfeld, J. F. A covalent fluorescent-gold immunoprobe: "simultaneous" detection of a pre-mRNA splicing factor by light and electron microscopy. J. Histochem. Cytochem., 45, 947-956 (1997).

    Reprint: http://www.jhc.org/cgi/reprint/45/7/947.pdf

  • Robinson, J. M., and Vandr, D. D. Efficient immunocytochemical labeling of leukocyte microtubules with FluoroNanogold: An important tool for correlative microscopy. J. Histochem. Cytochem., 45, 631-642 (1997).

    Reprint: http://www.jhc.org/cgi/reprint/45/5/631.pdf

    Takizawa, T.; Suzuki, K., and Robinson, J. M.: Correlative Microscopy Using FluoroNanogold on Ultrathin Cryosections: Proof of Principle; J. Histochem. Cytochem., 46, 1097-1102 (1998).

    Reprint: http://www.jhc.org/cgi/reprint/46/10/1097.pdf

Combined fluorescent and gold probes with other fluorophores are now under development for future commercial introduction.

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Other Recent Publications

Rajamannan and co-workers tried different methods of antigen retrieval to prepare specimens for studies on the localization of caveolin 1 in aortic valve endothelial cells with 15 to 40 nm gold; they found that SDS produced the most consistent labeling pattern, and quantitative evaluation revealed that SDS significantly increased the labeling density in Spurr-embedded cells.

Reference:

Rajamannan, N. M.; Springett, M. J.; Pederson, L. G., and Carmichael, S. W.: Localization of Caveolin 1 in Aortic Valve Endothelial Cells Using Antigen Retrieval. J. Histochem. Cytochem., 50, 617-628 (2002).

Abstract (Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/5/617

Sandhu and group, who have investigated a number of applications of 2-10 nm mixed monolayer-protected gold clusters (MMPCs), have described the use of gold nanoparticles functionalized with quaternary amines to transfect mammalian cells. The most efficient nanoparticle studied (MMPC 7) was approximately 8-fold more effective than 60 kDa polyethylenimine, a widely used transfection agent.

Reference:

Sandhu, K. K.; McIntosh, C. M.; Simard, J. M.; Smith S. W.; and Rotello V. M.: Gold nanoparticle-mediated transfection of mammalian cells. Bioconjug. Chem., 13, 3-6 (2002).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11792172&dopt=Abstract

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