Technical Help for Silver Enhancement

Updated: May 11, 2006 On this page Which silver enhancer should I use? Background Signal Other makers' probes Osmication Stains Dual Gold-Silver / DAB Staining In Situ: No Labeling! Note: GoldEnhance Technical Support Moved Technical support for gold enhancement has been expanded to include detailed responses to recent questions, and moved to a new page: http://www.nanoprobes.com/TechGE.html. Other Resources Nanoprobes e-Newsletter Try our e-mail newsletter for tips and tricks for better gold labeling, and to keep up with new applications. Guide to Gold Cluster Labeling Detailed description of gold cluster labeling, tips and tricks for successful conjugations, isolation of conjugates, and how to calculate labeling. MSA Microscopy Listserver and Archives (view or search). Confocal Listserver and Archive (SUNY at Buffalo). Tips and Tricks (from the University of Florida ICBR EM core lab).

Which one should I use?

It depends which considerations are more important to you. HQ silver is intended to give the most uniform particle size: it has a protective thickening agent (similar to the Danscher process) which has been found to moderate the silver enhancement process. Its pH is closer to neutral and its ionic strength is low, which some researchers believe improves ultrastructural preservation. LI silver is more convenient to use (it is a two-component reagent, while HQ Silver is a 3-bottle kit) and the reaction is slower, allowing more control. It is also more light-stable.

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Background Signal

General:

1. Ensure that all samples are thoroughly washed with ultrapure or deionized water before silver enhancement: halide or other silver-precipitating anions will cause a precipitate to form, which is itself visible in the EM and will nucleate additional background staining.
2. Wash samples with a chelating agent before silver enhancement. We usually use disodium EDTA (0.05 M, pH 4.6) on blots as the last wash before adding the silver enhancement reagent.
3. Clean glassware (for mixing reagents, handling specimens or storing silver enhancers) with Farmer's solution for at least 20-30 minutes. Farmer's solution consists of 9 parts of 10 % sodium thiosulfate with 1 part of 10 % potassium ferricyanide.
4. Use plastic or teflonized (not metal) forceps and tools for handling specimens.

There are two approaches to reducing background staining: (a) modify the experimental conditions before and during silver enhancement; or (b) improve the "stopping" of the silver enhancement reaction or apply back development after it is complete.

Reducing background during reaction

In experiments with the combined fluorescein and Nanogold® probe FluoroNanogold, it was found that washing with 0.02 M sodium citrate buffer, pH 7.0, before silver enhancement gives significantly lower backgrounds than many other treatments. This was found to be effective when the Danscher silver enhancer was used for development. When Nanoprobes HQ Silver was used, 0.02 M sodium citrate buffer at pH 3.5 immediately before silver enhancement gave lower background. In immunoblots, we find that 0.05 M disodium EDTA, pH 4.6, before silver enhancement also helps minimize background.

Reference:

• Powell, R. D.; Halsey, Carol 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).

Addition of a small amount of the detergent Tween-20 to the Nanogold® incubation buffer helps to prevent hydrophobic interactions, which can be another source of non-specific binding. If the sample can tolerate it, washing with 0.6 M triethylammonium bicarbonate buffer, in which Nanogold® is highly soluble, can also lower background. Preparation of triethylammonium bicarbonate buffer is described in the following reference:

• Safer, D.; Bolinger, L., and Leigh, J. S.; J. Inorg. Biochem., 26, 77 (1986).

"Stop" Reactions and back development:

Under most circumstances, repeated washing with deionized or distilled water will be sufficient to halt the silver enhancement process. However, it may continue within the specimen, and this can sometimes give over-development or a dark appearance in the light microscope. In this case, one of the following methods may be used to "stop" the silver enhancement reaction:

• 1% acetic acid.
• 1% acetic acid followed by photographic fixer (Agefix, Agfa-Gevaert, or Ilfospeed 200, Ilford).
• direct photo fix, using those just mentioned.
• brief rinse in 2.5% sodium chloride.
• 15-25% aqueous sodium thiosulfate plus 15% sodium sulfite.
• 1% acetic acid, washes in acetate buffer, toning in 0.05% HAuCl₄ 3-10 min, with excess silver removed with 3% sodium thiosulfate. We found that Nanogold-labeled proteins run on a polyacrylamide gel kept low backgrounds when stopped with 10% acetic acid with 10% glucose in water, as opposed to just a water stop.

• Hainfeld, J. F., and Powell, R. D.: Nanogold Technology: New Frontiers in Gold Labeling. Cell Vision, 4, 408-432 (1997).

  Complete paper (PDF) (courtesy of the Cell Vision).

In the event that the reaction has proceeded too far, it may be "back-developed" to remove the excess background staining by treatment with Farmer's solution (0.3 ml 7.5% potassium ferricyanide, 1.2 ml of 20% sodium thiosulfate, 60 ml water). Application of this solution briefly to your sample before gold toning may help to remove backgroud silver deposition.

Reference:

• Danscher, G.: Histochemistry, 71, 1-16 (1981).

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Other Manufacturers' Probes

Yes, absolutely. And silver enhancers from other manufacturers will also work on our probes (both Nanogold® and colloidal gold probes), too.

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Osmication

Osmium tetroxide is a powerful oxidising agent and can re-oxidise the metallic silver deposited during silver enhancement to silver (I); either the silver (I) is then removed by other reagents, or the oxidation exposes the metallic silver to other silver-dissolving reagents. This is usually a significant problem only when osmium tetroxide and uranyl acetate are used together; in the absence of uranyl acetate, the effect is much less and may usually be eliminated by reducing the osmium tetroxide concentration. To render deposited silver particles impervious to etching, use gold toning:

Lower Osmium Tetroxide Concentration:

Burry and co-workers have found that silver etching by osmium tetroxide may be greatly reduced by using 0.1 % osmium tetroxide instead of 1 %; this has been found to give similar levels of staining.

Reference:

• Burry, R.W.: Pre-embedding immunocytochemistry with silver-enhanced small gold particles. In Immunogold silver staining: Principles, methods and applications; M. A. Hayat (Ed.). CRC Press, Boca Raton, FL (1995), p. 217-230.

Gold toning prevents particle loss:

Gold toning is the post-treatment of silver enhanced immunogold particles with a reagent which deposits a thin layer of gold onto their surface, rendering them impervious to etching. Two procedures have been described: 1. Arai, et al. (Suggestion from the Microscopy Listserver)

From Diana Van Driel (Department of Opthalmology, Sydney University, Australia 2006):

I do a lot of Ag-Au for EM and initially had just this problem. After normal embedding of the Ag enhanced tissue, there would sometimes be beautiful Ag particles, sometimes remains of Ag with holes where the particles had been, sometimes nothing at all. The problem turned out to be the uranyl acetate treatment. It appears that uranyl acetate dissolves out the silver in some way. Not using uranyl acetate cured the problem, but obviously contrast was then hopeless. I now gold tone tissue - the Au-Ag-Au complex is completely stable in osmium and uranyl acetate. There is an increase in the contrast of the tissue, which may cause some people problems, especially if high resolution is needed, but the method really works.

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

1. Arai, R., et al.; Brain Res. Bull., 1992, 28, 343-345.
2. Arai, R., and Nagatsu, I. In Immunogold-Silver Staining: principles, Methods >and Applications; M. A. Hayat (Ed.), CRC Press, Boca raton. FL (1995) ch. 13, pp 209-216.

2. An alternative procedure has been reported by Sawada et al.:

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

Reference:

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

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Stains

Stains such as uranyl acetate and lead citrate must be used after silver enhancement. Metal ions-particularly transition metals and metal susceptible to redox reactions-can catalyze the silver enhancement reaction and result in background signal.

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Dual labeling Using Immunogold-Silver and DAB

DAB and other enzyme substrates are redox active compounds, and the enzymes themselves work by redox reactions. This means that the enzymes, DAB and other substrates, and their products may catalyze silver deposition. However, these two methods may be used for dual labeling: immunogold staining and silver enhancement should be completed first and all silver thoroughly removed by washing with deionized water, and the enzyme labeling and DAB chromogenic substrate treatment performed after silver enhancement is complete.

References:

1. Li, H.; Ohishi, H.; Kinoshita, A.; Shigemoto, R.; Nomura, S., and Mizuno, N.: Localization of a metabotropic glutamate receptor, mGluR7, in axon terminals of presumed nociceptive, primary afferent fibers in the superficial layers of the spinal dorsal horn: an electron microscope study in the rat. Neuroscience lett., 223, 153-156 (1997).
   • Abstract (courtesy of the Neuroscience Letters).

2. Salas, P. J. I.: Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells. J. Cell Biol., 146, 645-657 (1999).
   • Abstract (courtesy of the Journal of Cell Biology).

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In Situ Hybridization: No Labeling!

Several users report that silver enhancement of a specimen labeled by in situ hybridization gives no signal. We do not know why this occurs, but the following have been found to help:

• Use the Danscher formulation of silver enhancer instead of HQ silver or LI Silver. For how to make this, see: Danscher, G.; Histochemistry, 71, 81 (1981).
• Reduce the amountof glutaraldehyde used for fixation as far as possible; the amount of paraformaldehyde may be correspondingly increased.

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Any tips for other users? Things you'd like to see us make? Or, if you want to tell us your question or problem, E-mail us at tech@nanoprobes.com.