Technical Help for FluoroNanogold

Updated: May 12, 2002 On this page Fluorescence is weak Reducing fluorescence background Reducing gold/silver background 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).

My Fluorescence Signal is weak!

Note: FluoroNanogold conjugates are supplied at the same concentration as regular Nanogold® reagents, and colloidal gold preparations from other companies: about 0.08 mg/mL (80 micrograms/mL). Many commercial fluorescently labeled antibodies are supplied in concentrations around 1 mg/mL. If you observe only weak fluorescence, you may be using too dilute a solution of the reagent: try a 5-fold more concentrated solution.

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How do I Reduce Fluorescence Background?

This usually means that the intensity of the signal relative to the background (the "signal-to-noise" ratio) is too low. The most effective resolution is to increase the specific signal intensity, by using a higher concentration of the FluoroNanogold or a longer incubation time. It is also possible that since both the Nanogold® label and fluorescein have a hydrophobic character, that hydrophobic interactions with components of the specimen may be causing the background.

Reagent concentration: Try increasing the concentration of the FluoroNanogold reagent five-fold or ten-fold. Changes in the concentration of primary antibody may also affect the fluorescence signal; therefore, this should be tried at a lower or higher dilution.

Adjusting camera exposure: Manual control of exposure can greatly help in reducing apparent background. FluoroNanogold is frequently compared with commercially available fluorescently labeled IgG conjugates, which since they are larger and more highly labeled, give brighter fluorescence. If automatic exposure adjustment is allowed with FluoroNanogold-stained specimens, the greater exposure can lead to higher apparent backgrounds. Fixing camera exposure manually can be used to overcome this effect.

Reducing hydrophobic interactions

The following reagents may be added to the incubation or wash buffers to reduce hydrophobic interactions of the FluoroNanogold with cell or tissue components:

• Adding a small amount of 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling CO₂ into an aqueous suspension of triethylamine with stirring) to the wash buffer after FluoroNanogold incubation. Reference: Safer, D.; Bolinger, L., and Leigh, J. S.; J. Inorg. Biochem., 26, 77 (1986).
• Adding a small amount (0.1 % to 1 %) of detergent, such as Tween-20, or Triton X-100.
• Adding a small amount (0.1 % to 0.5 %) of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane; this may also help to eliminate interactions between the hydrophobic functional groups of the FluoroNanogold and hydrophobic regions in the specimen.

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How do I Reduce Silver/Gold Background?

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.

It is somewhat difficult to optimize the concentrations and buffers for FluoroNanogold since fluorescent reagents are usually used at higher concentrations than immunogold. The concentration of FluoroNanogold required in order to obtain both fluorescence and electron micrographs from one labeling procedure may be a compromise, and since the concentration of Nanogold® will be higher than that required only for Nanogold® labeling, this can lead to higher than normal backgrounds.

Controlling background using buffers and reaction conditions:

When FluoroNanogold was tested, we found that using a sodium citrate buffer as the final wash before silver enhancement gave a big improvement in background. Washing with 0.02 M sodium citrate, pH 7.0 immediately before silver enhancement resulted in lowest background staining when using the Danscher silver enhancement protocol, while 0.02 M sodium citrate, pH. 3.5, gave the best results with HQ Silver. In addition, in blots using LI Silver, 0.05 M disodium EDTA, pH 4.56, applied immediately before silver enhancement, also gave low background staining (it may chelate and remove extraneous metal ions which can act as nucleating sites for silver enhancements).

This is an excerpt from our paper in the Journal of Histochemistry and Cytochemistry (July 1997); this EM silver enhancement procedure was optimized by Dr. David L. Spector (Cold Spring Harbor Laboratory).

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).

Labeling of the SC35 pre-mRNA splicing factor in HeLa cells and Microscopy

HeLa cells were grown on coverslips for two days and fixed, washed and permeabilized as previously described (Spector et al. 1991). Next, the cells were incubated for one hour with antibody to SC35 (1:800 in PBS containing 0.1 % normal goat serum (NGS)), then washed in PBS containing 1 % NGS, then incubated with a 1 : 10 dilution of fluorescein/Nanogold-conjugated Fab' goat anti-mouse IgG (H+L) for 1 hour at room temperature. At this point, fluorescence pictures were obtained using a Nikon FXA microscope equipped with a 60X/1.3N.A. objective. After four ten-minute washes with PBS, the cells were fixed in 1 % glutaraldehyde in PBS for 15 minutes, followed by three ten-minute washes with PBS. Prior to silver enhancement, the buffer was changed to 0.02 M sodium citrate buffer, pH 7.0. The cells were extensively washed in this buffer (chlorides must be removed before silver enhancement). The silver enhancement procedure was then performed in the darkroom using a Thomas Duplex sodium vapor safelight.

HQ Silver (Nanoprobes, Incorporated, Stony Brook, NY) was used to enhance the gold probe in the cells. HQ Silver was prepared by vortexing a 1 : 1 : 1 mixture of initiator, moderator and activator. The backs of the coverslips were dried using filter paper and 200 microliters of the silver enhancement solution was applied to the cell side of the coverslip. After approximately 15 minutes, or when the silver changed from clear to gray, the silver was washed off the coverslip using citrate buffer. Once silver enhancement was complete, the cells were washed extensively with citrate buffer to remove any non-specific silver deposits and to prevent any further silver enhancement.

Following washes in citrate buffer, the cells were dehydrated through a graded series of ethanol. The cells were infiltrated with a 50 : 50 solution of ethanol and Epon-Araldite for 18 hours followed by 100 % Epon-Araldite for 8 hours. The coverslips were embedded in Epon-Araldite and placed in an oven at 60°C for 48 hours to polymerize. The glass coverslips were removed using hydrofluoric acid. Silver-enhanced gold-labeled sections were cut on a Reichert Ultracut E ultramicrotome using a Diatome diamond knife. Sections were picked up on 200 mesh copper grids and counterstained with 5 % uranyl acetate for 5 minutes and with Reynolds lead citrate for 1 minute. Some sections were also EDTA regressed for 30 minutes. Sections were viewed with a Hitachi H-7000 transmission electron microscope operated at 75 kV. Reference:

• Spector, D. L, Fu X.-D., and Maniatis, T. Associations between distinct mRNA splicing components and the cell nucleus. EMBO J. 10, 3467-3481 (1991).

"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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