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Updated: March 1, 2001

N A N O P R O B E S     E - N E W S

Vol. 2, No. 2          March 1, 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 [email protected].

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Combine Gold Labeling and Silver Enhancement with Other labels

You can combine Nanogold labeling and silver or gold enhancement with other labeling methods, such as enzymatic labeling, in the same specimen, and readily distinguish the two different types of signal. In these two papers, the investigators used Nanogold with silver enhancement to mark one site of interest, and then used peroxidase/DAB labeling to label a second site:

Bernard, V.; Levey, A. I., and Bloch, B.: J. Neurosci., 19, 10237-10249 (1999).

Li, H.; Ohishi, H.; Kinoshita, A.; Shigemoto, R.; Nomura, S., and Mizuno, N.: Neuroscience lett., 223, 153-156 (1997).

Salas, P. J. I.: J. Cell Biol., 146, 645-657 (1999).

Visit the references section of our web site to see some of the other applications of silver and gold enhancement. Like all our reference pages, the one for silver and gold enhancement is available sorted by product, or by application: (sorted by application). (sorted by product).

If you decide to try this procedure, we recommend completing the Nanogold labeling and silver or gold enhancement before the peroxidase/DAB labeling. This is because the reagents used with DAB/peroxidase staining can nucleate silver or gold deposition during enhancement; if you conduct this procedure before gold labeling and silver or gold enhancement, you may find non-specific background staining with the silver or gold enhancement.

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Gold Toning: How to Prevent Osmium Etching of Silver-Enhanced Gold

If you use osmium tetroxide after immunogold staining and silver enhancement, you might be etching the silver - reducing the size of the particles, or removing them entirely. To avoid this problem, you can either (a) reduce the concentration of osmium, (b) use gold toning after silver enhancement, or (c) enhance with gold instead of silver, using GoldEnhance.

If you are not using uranyl acetate, silver etching is rarely a problem. If it is, Burry and co-workers have found that it may be greatly reduced by using 0.1 % OsO4 instead of 1 %; osmication was performed for 30 minutes. This was found to give similar levels of staining to those found using 1 % osmium tetroxide, but 0.1 % OsO4 can be safely used after silver enhancement without gold toning. Reference:

Burry, R.W.:in M. A. Hayat ed. "Immunogold silver staining: Principles, methods and applications," CRC Press, Boca Raton, FL (1995).

If uranyl acetate staining is performed after osmication, the etching of silver is sometimes much worse, and the silver particles can be stripped away completely. We recommend gold toning if you intend to use osmication (at any concentration) and uranyl acetate. There are two procedures you can use:

  1. After silver enhancement, wash thoroughly with deionized water.
  2. 0.05 % gold chloride: 10 minutes at 4°C.
  3. Wash with deionized water.
  4. 0.5 % oxalic acid: 2 minutes at room temperature.
  5. 1 % sodium thiosulfate (freshly made) for 1 hour.
  6. Wash thoroughly with deionized water and embed according to usual procedure.

Arai, R., et al.; Brain Res. Bull., 28, 343-345 (1992).
Medline citation:

Arai, R., and Nagatsu, I.; in: "Immunogold-Silver Staining: principles, Methods and Applications;" M. A. Hayat, ed. Chap. 13, pp 209-216. CRC Press, Boca Raton. FL (1995).

Alternatively, this slightly gentler procedure has been reported:

  1. Rinse twice quickly in distilled water.
  2. 0.05 M sodium acetate (1 minute) then rinse again quickly.
  3. 0.05 % tetrachloroauric acid (2 minutes).
  4. Thorough rinsing in distilled water for 10 minutes, then osmicate.

Sawada, H., and Esaki, J.: Elect. Micro., 43, 361-66 (1994).
Medline citation:

The simplest remedy is to enhance with gold, using GoldEnhance. This is a new reagent developed by Nanoprobes that works like silver enhancement, except that it deposits gold instead of silver. GoldEnhance has other advantages, as well. The gold-enhanced particles are denser than silver-enhanced ones, and give superior back-scattered electron (BSE) detection in the scanning electron microscope (SEM). Unlike silver enhancement reagents, GoldEnhance may be used in the presence of chloride buffers without precipitation problems.

Technical help with silver enhancement:
Technical help with gold enhancement:
GoldEnhance: information and prices:
Our GoldEnhance paper from M & M 99:

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Make Your Own Double Labeled Probes

When you attach Nanogold to your probes, you are not restricted to *only* gold labeling. You can attach a second label to the probe, and then use the double-labeled probe to label your antigens for observation by two different, complementary imaging or detection modalities. Couple an enzyme as well as Nanogold and detect your antigens with chemiluminescent or chromogenic substrates. Use an amine-reactive fluorescent labeling reagent to put a novel fluorophore onto an antibody or protein that you have labeled with Monomaleimido-Nanogold. Once you have attached the gold, the link is stable, and you can couple a second label under a wide variety of reaction conditions.

Information and pricing:
Technical Help:

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Double labeling with Different Sized Golds

You can also double label with different sized gold particles, with or without silver enhancement. Takizawa and Robinson have demonstrated that the dense labeling of granules with Nanogold positive for DAF were easily distinguished from 10 nm colloidal gold labeled lactoferrin-containing granules; reference:

Takizawa, T. and Robinson, J.M.: J. Histochem. Cytochem., 42, 1615-1623 (1994).
Matsubara and co-workers stained GluR2/3 with dense fine deposits of silver enhanced Nanogold staining that were easily distinguishable from 30 nm colloidal gold immunostaining of glutamate. Nusser and colleagues used postembedding (freeze-substituted, Lowicryl embedded ultrathin sections) with silver amplified for 15 nm gold (giving large 40 nm particles), targeted to GABA, and silver intensified Nanogold, targeted to GABAA receptors, on the same section to simultaneously view the distributions of these targets in cerebellar granule cells. References:

Matsubara, A., J.H. Laake, S. Davanger, S.-I. Usami, and O.P. Otterson. 1996. J. Neuroscience, 16, 4457-4467.

Nusser, Z., J.D.B. Roberts, A. Baude, J.G. Richards, and P. Somogyi:The Journal of Neuroscience, 15, 2948-2960 (1995).

References for other applications of Nanogold:
Technical Help for Nanogold conjugates:

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Recent Papers

Dvorak and colleagues use used a pre-embedding immunonanogold ultra-structural method to define the subcellular distribution of a v-SNARE, VAMP, in three cell lineages (endothelial cell, pericyte, smooth muscle cell) in human skin venules and in two granulocyte lineages; staining was dense and specific in both skin and cell samples (see: Feng, D.; Flaumenhaft, R.; BandeiraMelo, C.; Weller, P. F., and Dvorak, A. M.: J. Histochem. Cytochem. 49: 293-304 (2001)).


For more details on the method, see: Feng, D.; Nagy, J. A.; Brekken, R. A.; Pettersson, A.; Manseau, E. J.; Pyne, K.; Mulligan, R.; Thorpe, P. E.; Dvorak, H. F., and Dvorak, A. M.: J. Histochem. Cytochem., 48, 545555 (2000).


Moise Bendayan has reviewed recent developments in the immunogold method (see: Bendayan, M.: Science, 291, 1363-1365 (2001)):

Abstract (requires registration):

Hong Yi and co-workers present a new double labeling method in the Journal of Histochemistry and Cytochemistry, in which both targets are labeled with ultrasmall gold, but distinguished by different lengths of silver enhancement (see: Yi, H.; Leunissen, J. L. M.; Shi, G.; Gutekunst, C.-A., and Hersch, S. M.: J. Histochem. Cytochem. 49: 279-284 (2001)).


Because Nanogold is based on a discrete molecule - a gold cluster complex - instead of a colloidal particle, it is highly monodisperse, and therefore well suited to this technique.

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