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Please note that our new, improved gold enhancement for EM - GoldEnhance™ EM Plus - has low background and slow development, solving the problems with the original version that are mentioned here.
- Guide to Gold Nanoparticle Labeling
Detailed description of gold nanoparticle labeling, tips and tricks for successful conjugations, isolation of conjugates, and how to calculate labeling.
I have heard about Gold Enhancement and was wondering if I could use it in my application. How is it different from silver enhancement, and are there any systems in which it should not be used?
Gold enhancement works very similarly to silver enhancement. In general, it may be used in place of silver enhancement in any system in which silver enhancement is currently used. it has several advantages which make it easier to use in some applications:
- Autonucleation is minimal even after 1-2 hours - more convenient for multiple samples, or if sample access is restricted (e.g. automated processing).
- May be safely used before osmium tetroxide staining without post-treatment such as gold toning.
- Improved backscatter detection of enlarged particles - more useful for SEM.
- Low viscosity for easy and accurate mixing of components.
- Gold Enhancement process is less pH dependent than silver enhancement - Goldenhance is supplied at neutral pH, and you can adjust pH for better sample preservation.
- Can be used in physiological buffers - gold is not precipitated as silver is (however, rinsing with water first is still recommended).
As far as we are aware, there are no silver enhancement applications in which GoldEnhance may not be used.
Catalog: GoldEnhance | References | Product Instructions
Please note that our new, improved version for EM - GoldEnhance™ EM Plus - has low background, which generally solves this problem.
When I perform gold enhancement, I see a non-specific "background" of fine gold particles. These are much smaller than the enlarged gold labels. How can I prevent this?
In some situations, GoldEnhance may produce a fine, "granular" non-specific background signal of small particles. There are several ways in which you can either slow down development so that you have more control over its progress, or chemically "stop" the development process. Which approach is best for the system under study depends upon whether you believe the primary problem is continued development after rinsing, in which case a chemical "stop" is appropriate, or excessively fast development before rinsing, in which case slowing down the reaction is most appropriate.
If you are observing fine background that seems to arise from continued development after rinsing to remove the GoldEnhance reagents, a number of "stop" procedures are available. The simplest and most universal is treatment with 1% or 2% freshly prepared sodium thiosulfate for a few seconds, and unless there are specific reasons that this would be harmful to your specimen, we usually suggest that you try this first.
A number of other specific methods have been reported in the literature for stopping silver enhancement. Since gold enhancement is a chemically similar process, these reactions are also applicable to gold enhancement:
- 1% acetic acid (Reference: Scopsi, L.: Silver-enhanced colloidal gold method. In: Colloidal Gold: Principles, Methods, and Applications; M. A. Hayat (Ed.), Vol. 1, p. 260. Academic Press, San Diego, CA (1989)).
- 1% acetic acid followed by photographic fixer (Agefix, Agfa-Gevaert, or Ilfospeed 200, Ilford) (Reference: Scopsi, L.: Silver-enhanced colloidal gold method. In: Colloidal Gold: Principles, Methods, and Applications; M. A. Hayat (Ed.), Vol. 1, p. 260. Academic Press, San Diego, CA (1989)).
- Direct photo fix, using those just mentioned (Reference: Burry, R. W.: Pre-embedding immunocytochemistry with silver-enhanced small gold particles, p. 217-230. In: Immunogold silver staining: Principles, methods and applications; M. A. Hayat (Ed.). CRC Press, Boca Raton, FL (1995)).
- Brief rinse in 2.5% sodium chloride (Reference: Scopsi, L.: Silver-enhanced colloidal gold method. In: Colloidal Gold: Principles, Methods, and Applications; M. A. Hayat (Ed.), Vol. 1, p. 260. Academic Press, San Diego, CA (1989)).
- 15-25% aqueous sodium thiosulfate plus 15% sodium sulfite.
Danscher, G.: Histochemical demonstration of heavy metals. A revised version of the silver sulphide method suitable for both light and electron microscopy. Histochemistry, 71, 1-16 (1981).
- 1% acetic acid, washes in acetate buffer, toning in 0.05% HAuCl4 for 3-10 minutes, with excess silver removed with 3% sodium thiosulfate. Nanogold-labeled proteins run on a polyacrylamide gel have been found to produce low backgrounds when stopped with 10% acetic acid with 10% glucose in water, compared with just water rinsing to stop development.
Takizawa, T., and Robinson, J. M.: Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections. J. Histochem. Cytochem., 42, 1615-1623 (1994).
If you believe that your principal problem with background is that the reaction is too fast and you need to slow down development, the following modifications are recommended. We suggest trying (1), (2) or (3) first:
Please note that our new, improved version for EM - GoldEnhance™ EM Plus - has low background and a slow development time, which solves this problem.
- Reduce the development time, to one minute. The specific signal may be sufficiently dense even at the shorter development times, so reducing the time further may remove the background while still giving a strong specific signal.
- When mixing and dispensing the GoldEnhance, use a mixture of 5 parts solution B (activator) to one part solution A (enhancer). Solution B contains a gold stabilizing agent to control the reactivity of gold in solution: by increasing the amount of B, you may inhibit the background while still permitting strong specific deposition.
- Substitute Solution D with 0.05M sodium phosphate with 0.1M sodium chloride, at pH 5.5. This will reduce the pH of the reaction mixture. Generally, autometallographic reactions (silver and gold enhancement) proceed more slowly at lower pH; b reducing the pH, you will slow the gold deposition reaction and gain more control.
- Increase the sodium chloride concentration in your substitute for D to 0.5M. We have occasionally observed that this reduces background (possibly by shortening the range of ionic interactions in the solution through higher ionic strength).
- Treat samples with Lugol's iodine (Sigma: 30 seconds) followed by 1% sodium thiosulfate (30 seconds) before application of the antibodies- this reagent is used to remove heavy metals from tissue slides for light microscopy, and since these can be catalysts for autometallographic development, this eliminates one source of background. We have found it to be essential for in situ hybridization detection with Nanogold and GoldEnhance. In the in situ hybridization procedure it is applied early, before the Nanogold reagent, so if it is possible it may be best to try it before adding the antibodies.
- Add a viscosity modifier, such as polyethylene glycol (carbowax, or one of the higher MW polethylene glycols, would be best: dissolve 4% in your substitute solution D to give 1% in the final mixture). Gum Arabic, if available, is also highly effective in silver enhancement solutions, although it must be allowed to dissolve for 24 hours before use: use 30% in solution D (make up a 60% solution, then mix with an equal volume of a double concentration of your substitute solution D). These slow down development in silver enhancement to give a very controlled, homogeneous rate of reaction, resulting in uniform particle size and morphology.
- Add a small amount of detergent, such as 0.1% Tween-20 (add as 0.4% in your substitute D to give 0.1% in the final solution). Detergents are known to modify the growth of nanoparticles, and act to negate hydrophobic interactions which can initiate background deposition, or facilitate the deposition of any pre-formed gold clusters from solution.
- Wash with 0.6 M triethylammonium bicarbonate buffer in 20 % isopropanol/water after gold enhancement. We find this buffer to be highly effective for dissolving small gold particles such as Nanogold, and it will help to remove any particulate deposits. It is prepared by bubbling carbon dioxide through a mixture of degassed water and degassed triethylamine. We recommend making a 2 M stock solution: dilute in water/isopropanol to give 0.6 M in 20 % isopropanol/water.
Reference for preparation:
Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77-91 (1986).
Dr. Wanzhong He, while he was working in the laboratory of Dr. Pamela J. Bjorkman in the Division of Biology at the California Institute of Technology was kind enough to send us feedback on on the results of applying these methods. A combination of steps was used to eliminate background:
- A ratio of one part solution A (enhancer) : 5 parts solution B (activator) was used.
- Solution D (buffer) was substituted with 0.05 M sodium phosphate with 0.1 M sodium chloride, pH 5.5.
- Enhancement was performed for 10-15 minutes rather than 3 minutes.
- The reaction was "stopped" using 3% Na2S2O3 for 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes.
Some preliminary images are shown below:
|left: Gold enhancement of intestinal tissue with Nanogold staining. (a) Nanogold particles, then enhanced with GoldEnhance EM for 3 minutes; (b) control with Nanogold labeling omitted, showing the granular background. right: analogous procedure, using modified protocol to control background, comprising: one part A (enhancer) : 5 parts B (activator); D (buffer) was substituted with 0.05M sodium phosphate with 0.1M sodium chloride, at pH 5.5; enhancement was performed for 10-15 minutes rather than 3 minutes, "stopped" using 3% Na2S2O3 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes. (a) Nanogold particles, enhanced with GoldEnhance EM; (b) control with Nanogold labeling omitted, identical gold enhancement procedure. Insets show experimental (left) and control (right) specimens (Thanks to Wanzhong He and Dr. Pamela Bjorkman for the images).
If none of these are effective, try our new, improved gold enhancer, GoldEnhance™ EM Plus. Or you might consider switching to silver enhancement using LI Silver or HQ Silver instead. If you are concerned about osmium etching of deposited silver, it is worth noting that Burry and co-workers find that this may frequently be avoided by using 0.1 % osmium tetroxide; this still gives excellent staining, but etching is no longer a problem (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 p. 217-230 (1995)).
Catalog: GoldEnhance | References | Product Instructions
Please note that our new, improved version for EM - GoldEnhance™ EM Plus - has a slow development time, which solves this problem.
I only gold enhance for three to five minutes - a much shorter time than silver enhancement - but the enlarged particles are already too large and obscure the structure of the specimen. How can I control the reaction so that the particles only grow as large as I need?
Depending on the sample properties and experimental conditions, the rate of gold particle enlargement with GoldEnhance can vary. In some systems, very rapid development of larger particles has been reported. If you observe the rapid formation of larger particles within a few minutes, we recommend the following:
- Examine after a shorter development time. In some applications, 1 or 2 minutes may be enough to enlarge the particles to the desired size.
- Substitute Solution D of the GoldEnhance with 0.05M sodium phosphate with 0.1M sodium chloride, adjusted to pH 5.5. This will reduce the pH of the reaction mixture, which will serve to slow down the rate of the gold deposition reaction.
- Increase the sodium chloride concentration in your substitute for D to 0.5M. We have occasionally observed that higher ionic strengths reduces background, and may act to moderate the rate of development (possibly by shortening the range of ionic interactions in the solution).
- Add a viscosity modifier, such as polyethylene glycol (PEG). A solution of one of the higher molecular weight forms, such as 1% carbowax (molecular weight 20,000), is best; dissolve 4% in solution D or your substitute; gum arabic, although it is somewhat less convenient to prepare since it requires longer to dissolve, is also effective. These are used to slow down development in silver enhancement and lead to uniform particle size and morphology, and it is reasonable to expect the same result with gold enhancement.
- Adding a small amount of detergent, such as 0.1% Tween-20 (add as 0.4% in solution D or your substitute). Detergents are known to modify the growth of gold nanoparticles and may provide a growth constraint during gold enhancement.
The most efficient strategy is to start with the first two suggestions; if these do not give the desired results, work through the other three, (3), (4) and (5) in the order given.
Catalog: GoldEnhance | References | Product Instructions
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