Updated: May 2, 2001
N A N O P R O B E S E - N E W S
Vol. 2, No. 4 May 2, 2001
This monthly newsletter is 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 tech@nanoprobes.com.
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If you are concerned about low abundance of your target, or have experienced low labeling, whether nucleic acid or antigen, you can increase the labeling by using a signal amplification procedure such as NEN's Tyramide Signal Amplification process. This has been highly effective for in situ hybridization detection:
Hacker, G. W., Hauser-Kronberger, C., Zehbe, I., Su, H., Schiechl, A., Dietze, O., and Tubbs, R.: In Situ localization of DNA and RNA sequences: Super-sensitive In Situ hybridization using Streptavidin-Nanogold®-Silver Staining: Minireview, Protocols and Possible Applications. Cell Vision, 4, 54-65 (1997).
However, you can also use this method to amplify cellular antigens:
Weipoltshammer, K.; Schofer, C.; Almeder, M., and Wachtler, F.: Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography. Histochem. Cell Biol., 114, 489-495 (2000).
Nanogold conjugates can also be used to detect products amplified by PCR:
Hacker, G. W.; Zehbe, I.; Hainfeld, J.; Sällström, J.; Hauser-Kronberger, C.; Graf, A.-H.; Su, H.; Dietze, O., and Bagasra, O; High-Performance Nanogold® In Situ Hybridization and In Situ PCR. Cell Vision, 3, 209 (1996).
For more information about the use of Nanogold in these applications:
Application - In situ hybridization: www.nanoprobes.com/InSitu.html
References for Nanogold labeling: www.nanoprobes.com/RefTopNG.html
Catalog information on Nanogold conjugates: www.nanoprobes.com/NanoAb.html
Tyramide Signal Amplification (TSA®), or Catalyzed reporter deposition (CARD), was developed by Dr. Mark BOBROW et al. (J. Immunol. Meth. 1989;124:279-85), is patented by NEN® Life Sciences Products (Boston, MA, USA; Web: http://www.nenlifesci.com).
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FluoroNanogold is a unique probe containing both Nanogold® and fluorescein. You can use it for truly correlative labeling on the cellular and macromolecular level, or for checking your labeling by fluorescence before EM processing.
Catalog information about FluoroNanogold: www.nanoprobes.com/FluoroN.html
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. For tips and tricks on FluoroNanogold labeling, and how to obtain the cleanest signal, visit our technical help page:
Technical help for FluoroNanogold: www.nanoprobes.com/TechFN.html
Look out for combined fluorescent and gold probes with other fluorophores: when these are available, we will announce them on our web site (www.nanoprobes.com).
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
Demonstration of correlative labeling:
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
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Because Nanogold® and undecagold are chemically cross-linked, like fluorescent labels, rather than adsorbed like colloidal gold, you can conjugate them to a wide variety of other molecules in addition to antibodies and proteins. Many other molecules that cannot be labeled with conventional colloidal gold can readily be labeled with Nanogold. Large or small, water-soluble or best handled in other solvents, all that is needed for successful gold-labeling is an accessible thiol (sulfhydryl) or amino- group. And with Monoamino-Nanogold, you can use many other heterobifunctional cross-linking reagents to link Nanogold to other functional groups.
Some examples:
Undecagold can even be used as an isomorphous heavy atom replacement to derivatize proteins for X-ray crystallographic studies:
References: www.nanoprobes.com/RefTopUG.html#Uiso
How do you get the best results from labeling, and how do you know whether it worked? Our Guide to Gold Cluster Labeling shows you the process step-by-step, with all the factors that can affect your labeling reaction described and explained. The Guide also includes the UV/visible spectra of Nanogold and undecagold both unconjugated and conjugated, and shows you how to calculate your labeling exactly.
Guide to Gold Cluster Labeling: www.nanoprobes.com/LGuide.html
Spectra and labeling calculations: www.nanoprobes.com/LGuide4.html
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If you'd like to avoid problems with your samples charging under the electron beam, consider conductive polymer substrates, prepared with 3-octadecylpyrrole and 3-octadecanoylpyrrole. These are used with ferric chloride as a catalyst and a trace of pyrrole vapor to form thin layers of conductive polypyrroles in a Langmuir-Blodgett trough or similar apparatus, which can then be transferred to EM specimens.
For more details, see our paper: www.nanoprobes.com/Conduct.html
Catalog information: conductive polymers: www.nanoprobes.com/Polyms.html
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Kv4.2 subunit, a member of K+channel gene family, is considered to play a major role in the formation of depolarization-activated transient outward K+ current channels in the mammalian heart. Takeuchi and co-workers used correlative fluorescence and electron microscopy with FluoroNanogold probes to investigate the subcellular localization of Kv4.2 subunit in the rat heart, and confirmed its distribution and role:
Takeuchi, S.; Takagishi, Y.; Yasui, K.; Murata, Y.; Toyama, J., and Kodama, I.: Voltage-gated K(+)Channel, kv4.2, localizes predominantly to the transverse-axial tubular system of the rat myocyte. J. Mol. Cell.Cardiol., 32, 1361-1369 (2000).
Abstract: http://www.idealibrary.com/links/citation/0022-2828/32/1361
Qualmann, Kessels et al studied the effects of an estradiol hormone pulse on the localization and estradiol receptor content and localization of lysosomes in the porcine endometrium, using Nanogold-Fab' fragments in a post-embedding immunolabeling procedure to show that the number of lysosomes near the nucleus, and their immunostaining for estradiol receptor a, increased after the pulse, suggesting a role for lysosomes in the degradation of receptor shuttling out of the nucleus:
Qualmann, B.; Kessels, M. M.; Thole, H. H., and Sierralta, W. D.; A hormone pulse induces transient changes in the subcellular distribution and leads to a lysosomal accumulation of the estradiol receptor alpha in target tissues. Eur. J. Cell Biol., 79, 383-93 (2000).
Reprint: http://www.urbanfischer.de/journals/ejcb/content/2000/issue6/2510061a.pdf
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