Updated: August 16, 2002

N A N O P R O B E S E - N E W S

Vol. 3, No. 7 August 13, 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 tech@nanoprobes.com.

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Chemically Selective Labeling: Localizing Cysteines and Disulphide Bonds During Spermiogenesis

Gimenez-Bonafe and co-workers recently used gold labeling with Monomaleimido Nanogold® to localize and quantitate interprotamine disulfide bonds during spermiogenesis: they used the disappearance of Nanogold labeling as an indicator of the formation of disulfide bonds by cysteine residues. Pieces of gonads or sperm pellets were fixed in 4 % paraformaldehyde / 0.2 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, then embedded in Lowicryl resin. The grids were incubated with 12 nmol/mL of Monomaleimido Nanogold in PBS for 30 minutes; after washing several times with distilled water, they were developed with HQ Silver for observation in the electron microscope.

During the first step of spermiogenesis, protamines are found spread over very slightly condensed chromatin with their cysteines in a reactive state (protamine-cys-SH). From this stage the interprotamine disulfide bonds are established in a progressive way. First they are formed inside the chromatin fibers. Subsequently, they participate in the mechanism of fiber coalescence and finally, in the last step of spermiogenesis, the remaining free reactive -SH groups of cysteine form disulfide bonds, thus promoting a definitive stabilization of the nucleoprotein complex in the ripe sperm nucleus.

Reference:

Gimenez-Bonafe, P.; Ribes, E.; Sautière, P.; Gonzalez, A.; Kasinsky, H.; Kouach, M.; Sautière, P.-E.; Ausio, J., and Chiva, M.: Chromatin condensation, cysteine-rich protamine, and establishment of disulphide interprotamine bonds during spermiogenesis of Eledone cirrhosa (Cephalopoda). Eur. J. Cell Biol., 81, 341-349 (2002).

Abstract (MedLine):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12113475&dopt=Abstract

The different reactivities available with Nanogold provide several options for chemically specific labeling. For example, you can use Mono-Sulfo-NHS-Nanogold to selectively label amines, such as lysine residues; or Monoamino Nanogold to label activated carboxylic acids, or aldehydes.

More information on Nanogold labeling:

Nanogold labeling reagents - catalog info: http://www.nanoprobes.com/LabRgts.html
Monomaleimido Nanogold - product info: http://www.nanoprobes.com/Inf2020.html
Mono-Sulfo-NHS-Nanogold - product info: http://www.nanoprobes.com/Inf2025.html
Monoamino Nanogold - product info: http://www.nanoprobes.com/Inf2021.html
Nanogold labeling reagents - Tech Help: http://www.nanoprobes.com/TechNGlr.html
Guide to Gold Cluster Labeling: http://www.nanoprobes.com/LGuide.html

Also, visit our applications page to see some of the other applications of these reagents:

http://www.nanoprobes.com/Applic.html

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Getting the Best Results with Fluoronanogold - Update

We are currently developing new conjugates to expand our line of FluoroNanogold probes, unique reagents containing both Nanogold® and a fluorescent group, which may be used for correlative fluorescence and electron microscopy labeling or for checking labeling by fluorescence before EM processing. One of the challenges we address in developing these probes is to reduce non-specific binding; this is an important issue because FluoroNanogold combines two labels that are frequently used in different concentrations under different conditions, so compromise and optimization may be required to obtain the best performance. We have found that the following methods may help to reduce background:

The most effective blocking agent we have tested during our recent experiments is 5 % nonfat dried milk. This was found to be particularly effective when mixed with and added to the specimen together with the FluoroNanogold conjugate. Cold-water fish gelatin has also been found to be helpful for gold probes generally.
Adjusting camera exposure: manual control of exposure can also help in reducing apparent background. FluoroNanogold is frequently compared with commercially available fluorescently labeled IgG conjugates. Since these are larger and more highly labeled, they give brighter fluorescence. If automatic exposure adjustment is allowed with FluoroNanogold-stained specimens, the greater exposure can lead to higher apparent backgrounds. Setting the camera exposure manually can be used to overcome this effect.
For reducing the background in electron microscopy, sodium citrate buffer was found to be more effective than other buffers when used as a wash before silver enhancement. 0.02 M sodium citrate at pH 7.0 works well with HQ Silver, while pH 3.5 works best with the Danscher silver formulation (see Powell et al, 1997, reference below).
Background binding is often attributed to hydrophobic interactions (both the gold and fluorescent labels have some hydrophobicity), and therefore adding reagents that reduce hydrophobic interactions to the wash buffer may help remove non-specific binding. Examples include 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring. Reference: Safer, D.; Bolinger, L., and Leigh, J. S.; J. Inorg. Biochem., 26, 77 (1986); 0.1 % to 1 % detergent, such as Tween-20, or Triton X-100; and 0.1 % to 0.5 % of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.

More information:

Catalog information: http://www.nanoprobes.com/FluoroN.html
Technical help for FluoroNanogold: http://www.nanoprobes.com/TechFN.html
References for FluoroNanogold: http://www.nanoprobes.com/RefTopFN.html

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 Vandré, 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

Note: we do not offer tracers or other probes in which fluorescent groups are linked directly to gold particles, because such probes would not fluoresce! Gold particles absorb strongly at the emission wavelengths of commonly used fluorophores, and are therefore good acceptors for resonance energy transfer. This means that were the fluorophore conjugated directly to the gold particle, the fluorescence would be quenched. We have described this process in our 1998 paper (Powell, R. D.; Halsey, C. M. R., and Hainfeld, J. F.: Combined fluorescent and gold immunoprobes: Reagents and methods for correlative light and electron microscopy. Microsc. Res. Tech., 42, 2-12 (1998)).

To make sure fluorescence is preserved, the gold particle and fluorophore need to be spaced several nanometers apart, as they are in FluoroNanogold antibody conjugates. The best approach to preparing such probes is to separately attach the gold and fluorescent groups to a larger molecule.

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Gold-Facilitated In Situ Hybridization has High Interobserver Reproducibility

We recently described 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; by controlling the sizes of the signals, interpretation is simplified, as it now depends upon the overall staining pattern rather than spot counting. An interobserver reproducibility study among five practicing pathologists has now shown that interpretation of this assay is highly reproducible. The average kappa among GOLDFISH observers was 0.84, which was at least or concordant of observers scoring nuclear grade (kappa = 0.50) and the presence of in situ carcinoma (kappa = 0.57) by conventional histopathology.

Reference:

Tubbs, R.; Skacel, M.; Pettay, J.; Powell, R.; Myles, J.; Hicks, D.; Sreenan, J.; Roche, P.; Stoler, M. H., and Hainfeld, J.: Interobserver Interpretative Reproducibility of GOLDFISH, A First Generation Gold-Facilitated Auto-metallographic Bright Field In Situ Hybridization Assay for HER-2/neu Amplification in Invasive Mammary Carcinoma. Am. J. Surg. Pathol., 26, 908-913 (2002).

Abstract (courtesy of Lippincott, Williams and Wilkins): http://ipsapp003.lwwonline.com/content/getfile/2302/32/9/abstract.htm

This method has a number of advantages over fluorescence in situ hybridization (FISH):

Simultaneous visualization of the underlying morphology.
Black, punctate stain is easily distinguished from other stains.
Staining is permanent, and does not photobleach.
Oil immersion not required.
Higher resolution is also suitable for electron microscopy.

More information:

About Nanogold-ISH detection: http://www.nanoprobes.com/InSitu.html
Other references: http://www.nanoprobes.com/RefTopNG.html#Ninsitu
Current research protocols (G. W. Hacker): http://www.sbg.ac.at/kgg/protocols/protocols.htm
Product Information - Nanogold-Streptavidin: http://www.nanoprobes.com/Inf2016.html
Gold enhancement: http://www.nanoprobes.com/GoldEnhance.html

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New Book Provides Detailed Protocols; Other Help and Support

A new reference book on immunogold-silver staining is now available, including three chapters written by Nanoprobes scientists which include detailed descriptions of many of the methods and protocols used most effectively with 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.

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

On our web site, we also provide detailed technical help for all our products, as FAQs; most common problems or questions are answered here. We also include a Guide to Gold Cluster Labeling that discusses how to optimize labeling, separate conjugates, and calculate the extent of labeling.

Technical Help index: http://www.nanoprobes.com/Tech.html
Guide to Gold Cluster Labeling - overview: http://www.nanoprobes.com/LGuide.html

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New at Nanoprobes

Nanoprobes recently received a new Phase I SBIR grant from the National Cancer Institute (National Institutes of Health) to develop novel metal nanoparticle reagents and conjugates as improved contrast agents for magnetic resonance imaging. We also welcomed two new staff in the last month: Theresa Focella, who joins us to help with this and other research projects, and Claudia Gonzalez, who will be part of our manufacturing and product development team.

Details and other Nanoprobes News: http://www.nanoprobes.com/News.html
Information about our Company: http://www.nanoprobes.com/Company.html

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Other Recent Publications

To make in situ hybridization more consistently successful, Clive Taylor and colleagues have investigated the application of antigen retrieval conditions - heating and pH - to the retrieval of DNA in archived formalin-fixed paraffin-embedded tissue specimens. Generally, the best retrieval was found near pH 9 and 120°C.

Reference:

Shi, S. R.; Cote, R. J.; Wu, L.; Liu, C.; Datar, R.; Shi, Y.; Liu, D.; Lim, H., and Taylor, C. R.: DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH. J. Histochem. Cytochem., 50, 1005-1011 (2002).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/8/1005

Meanwhile, Ooorschot and co-workers describe a method for preparing ultrathin cryosections that allows visualization of the cells in their in situ orientation, enabling the study of polarized cells and facilitating correlative light and electron microscopy. Cultured cells are sampled from a petri dish or coverslip by embedding them in a 12% gelatin slab; subsequently, sections can be prepared in parallel or perpendicular to the plane of growth.

Reference:

Oorschot, V.; Heidi de Wit, H.; Annaert, W. B., and Klumperman, J.: A Novel Flat-embedding Method to Prepare Ultrathin Cryosections from Cultured Cells in Their In Situ Orientation. J. Histochem. Cytochem., 50, 1067-1080 (2002).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/8/1067

Iborra has used colloidal gold labeling to study gene expression in situ, and recently reviews some of this work in an article describing the path RNA takes from the nucleus to the cytoplasm, and the distribution of the active sites for the various processes that it undergoes during its journey.

Reference:

Iborra, F. J.: The path that RNA takes from the nucleus to the cytoplasm: a trip with some surprises. Histochem. Cell Biol., 118, 95-103 (2002).

Abstract (courtesy of Histochemistry and Cell Biology):
http://link.springer.de/link/service/journals/00418/contents/02/00441/

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