FLUORONANOGOLD^TM** PRODUCT INFORMATION

Alexa Fluor®* 488-FluoroNanogold^TM**-Fab'

* **	Alexa Fluor is a registered trademark of Molecular Probes, Inc. Patented technology.

Contents

1. Product Information
2. General Considerations for Immunostaining with Alexa Fluor® FluoroNanogold^TM Reagents
3. Using Stains with Alexa Fluor® FluoroNanogold^TM
4. Temperature Caution
5. Thiol Caution
6. Fluorescence Microscopy with Alexa Fluor® FluoroNanogold^TM
7. Electron Microscopy Immunolabeling with Alexa Fluor® FluoroNanogold^TM
   1. Cells in Suspension
   2. Thin Sections
8. Special Considerations for Viewing Alexa Fluor® FluoroNanogold^TM in the Electron Microscope
9. Silver Enhancement of Alexa Fluor® FluoroNanogold^TM for EM
10. Gold Enhancement of Alexa Fluor® FluoroNanogold^TM for EM
11. Immunoblotting
12. References

Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Non radioactive and non carcinogenic.

Product Information

The absorbtion maximum of Alexa Fluor®-488 occurs near 494 nm, and the emission maximum is near 519 nm. These values are very similar to those of fluorescein (green), so a fluorescein filter set is recommended for fluorescence observation.

Figure 1: Fab' covalently conjugated to Alexa Fluor® 488 and NANOGOLD® to give Alexa Fluor®-FluoroNanogold^TM.

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General Considerations for Immunostaining with FluoroNanogold^TM Reagents

The major difference will be in the results:

• Alexa Fluor®-FluoroNanogold^TM contains an extremely uniform 1.4 nm diameter gold particle (±10%).

• Fab-Alexa Fluor®-FluoroNanogold^TM is smaller than a single whole IgG molecule. It is not significantly larger than Fab'-NANOGOLD®, the smallest gold immunoprobe commercially available, and will penetrate and reach antigens inaccessible to other gold probes.

• Fab-Alexa Fluor®-FluoroNanogold^TM is chromatographically purified through gel filtration columns. There are absolutely no aggregates or other molecular weight impurities. This is in sharp contrast to colloidal gold conjugates which usually are prepared by centrifugation to remove the largest aggregates, and frequently contain smaller aggregates.

• Close to 1 NANOGOLD® label to 1 Fab' (or IgG) make this product distinct from the 0.2 - 10 variable stoichiometry of other colloidal gold antibody preparations.

• Alexa Fluor®-FluoroNanogold^TM particles do not have affinity to proteins as do colloidal golds. This reduces background and false labeling.

• Alexa Fluor®-FluoroNanogold^TM develops better with silver than do most colloidal golds, giving it higher sensitivity. Silver enhancement can be used to make the immunolabeling useful for electron microscopy, light microscopy, and immunoblotting with improved results.

Using EM Stains with FluoroNanogold^TM

1. Use of reduced amounts or concentrations of usual stains.
2. Use of lower atomic number stains such as NANOVAN^TM, a Vanadium based stain.⁵
3. Enhancement of FluoroNanogold^TM with silver developers, such as LI SILVER or HQ SILVER.
4. Enhancement of Alexa Fluor®-FluoroNanogold^TM with the gold developer, GOLDENHANCE.

Thiol Caution

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Temperature caution

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Methods

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Fluorescence Microscopy Immunolabeling with Alexa Fluor®-FluoroNanogold^TM

The procedure below³ describes an example of the use of a Alexa Fluor®-FluoroNanogold^TM conjugate as a secondary antibody probe. Dilutions of Alexa Fluor®-FluoroNanogold^TM will vary with different procedures, but a 5-fold or 10-fold dilution is advisable as a starting point for most applications; for simultaneous electron microscopy labeling, a compromise between the optimum concentrations for fluorescence and electron microscopy maybe necessary. Other protocols and techniques used with fluorescently-labeled antibodies may also be used with Alexa Fluor®-FluoroNanogold^TM conjugates.

1. Fix cells in freshly-prepared 2 % formaldehyde in PBS for 15 mins at 20°C; alternatively, fix in 100 % methanol at -20°C for 3 minutes; if methanol fixation is used, skip to step 4.
2. Wash in PBS (3 x 10 mins).
3. Permeabilize in 0.2 % Triton X-100 plus 1 % normal serum (NS) from the host species of the Alexa Fluor®-FluoroNanogold^TM-conjugated antibody in PBS at pH 7.3 for 5 minutes on ice.
4. Wash in PBS with 1 % NS (3 X 10 mins).
5. Incubate in the appropriate concentration of primary antibody for 1 hour at room temperature in a humidified chamber. If using 22 mm X 22 mm square cover slips, 30 microliters of diluted antibody is placed on the coverslip and the coverslip is inverted onto a glass slide. The slide is then placed in a humidified chamber which is incubated at room temperature.
6. Wash in PBS with 1 % NS + 5 % non-fat dried milk (3 X 10 mins).
7. Incubate with Alexa Fluor®-FluoroNanogold^TM reagent at a dilution of 1 : 5 to 1 : 10 (diluted in buffer containing 5 % non-fat dried milk) for 1 hour in a humidified chamber at room temperature.
8. Wash in PBS (4 X 10 mins).
9. Mount coverslip with a drop of mounting medium. Observe as usual.

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Electron Microscopy with Alexa Fluor®-FluoroNanogold^TM

If aldehyde-containing reagents have been used for fixation, these must be quenched before labeling. This may be achieved by incubating the specimens for 5 minutes in 50 mM glycine solution in PBS (pH 7.4). Ammonium chloride (50 mM) or sodium borohydride (0.5 - 1 mg/ml) in PBS may be used instead of glycine.

Cells in Suspension

If the cells are already labeled, mount, stain and observe as usual. If a different specimen is to be used, the procedure below is recommended:

1. Optional fixing of cells: e.g., with glutaraldehyde (0.05 - 1% for 15 minutes) in PBS. Do not use Tris buffer since this contains an amine. After fixation, centrifuge cells (e.g. 1 ml at 10,000,000 cells/ml) at 300 X g, 5 minutes; discard supernatant; resuspend in 1 ml buffer. Repeat this washing (centrifugation and resuspension) 2 times.
2. Incubate cells with 0.02 M glycine in PBS (5 mins). Centrifuge, then resuspend cells in PBS-BSA buffer (specified below) for 5 minutes.
3. Place 50 - 200 microliters of cells into Eppendorf tube and add 5 - 10 microliters of primary antibody (or antiserum). Incubate 30 minutes with occasional shaking (do not create bubbles which will denature proteins).
4. Wash cells using PBS-Milk as described in step 2 (2 X 5 mins). Resuspend in 1 ml PBS-Milk buffer.
5. Dilute Alexa Fluor®-FluoroNanogold^TM ~ 5 to 10 times in PBS-Milk buffer and add 30 ml to cells; incubate for 30 minutes with occasional shaking.
6. Wash cells in PBS-BSA buffer as described in step 1 (2 X 5 mins).
7. Fix cells and antibodies using a final concentration of 1% glutaraldehyde in PBS for 15 minutes. Then remove fixative by washing with PBS buffer (3 X 5 mins).

PBS-Milk Buffer: 20 mM phosphate 150 mM NaCl pH 7.40 5% Non-fat dried milk (final concentration) Optional, may reduce background: 0.5 M NaCl 0.05% Tween 20 0.1% gelatin (high purity)

PBS Buffer: 20 mM phosphate 150 mM NaCl pH 7.40

Negative staining may be used for electron microscopy of small structures or single molecules which are not embedded. Negative stain must be applied after the silver enhancement. NANOVAN^TM negative stain is specially formulated for use with NANOGOLD® reagents;⁵ it is based on vanadium, which gives a lighter stain than uranium, lead or tungsten-based negative stains and allows easier visualization of Alexa Fluor®-FluoroNanogold^TM particles with little or no silver enhancement.

Thin Sections

Labeling with Alexa Fluor®-FluoroNanogold^TM may be performed before (the pre-embedding method)^8,9 or after embedding and sectioning (the post-embedding method).^8,9 The procedures for both methods are described below.

Thin sections mounted on grids are floated on drops of solutions on parafilm or in well plates. Hydrophobic resins usually require pre-etching.

PROCEDURE FOR PRE-EMBEDDING METHOD:⁸

If specimen has already been labeled with Alexa Fluor®-FluoroNanogold^TM, skip to step 9. If a fresh specimen is required for EM, the following procedure is recommended.

1. Float on a drop of water for 5 - 10 minutes.
2. Incubate cells with 1 % bovine serum albumin in PBS buffer at pH 7.4 for 5 minutes; this blocks any non-specific protein binding sites and minimizes non-specific antibody binding.
3. Incubate with primary antibody, diluted at usual working concentration in PBS-milk buffer or PBS containing 1 % BSA (1 hour or usual time. Buffer formulations are given below).
4. Rinse with PBS-Milk (3 X 1 min).
5. Incubate with Alexa Fluor®-FluoroNanogold^TM reagent diluted 1/5 - 1/20 in PBS- Milk with 1 % normal serum from the same species as the Alexa Fluor®-FluoroNanogold^TM reagent, for 10 minutes to 1 hour at room temperature.
6. Rinse with PBS- Milk (3 X 1 min), then PBS (3 X 1 min).
7. Postfix with 1 % glutaraldehyde in PBS (10 mins).
8. Rinse in deionized water (2 X 5 min).
9. Perform silver or gold enhancement (e.g., HQ Silver or GoldEnhance), as specified in those product directions.
10. Dehydrate and embed according to usual procedure.
11. Stain (uranyl acetate, lead citrate or other positive staining reagent) as usual before examination.

PROCEDURE FOR POST-EMBEDDING METHOD:⁸

1. Prepare sections on plastic or carbon-coated nickel grid. Float on a drop of water for 5 - 10 minutes.
2. Incubate with 1 % solution of bovine serum albumin in PBS buffer at pH 7.4 for 5 minutes to block non-specific protein binding sites.
3. Incubate with primary antibody, diluted at usual working concentration in PBS containing 1 % BSA (1 hour or usual time).
4. Rinse with PBS-Milk (3 X 1 min).
5. Incubate with Alexa Fluor®-FluoroNanogold^TM reagent diluted 1/5 - 1/20 in PBS- Milk with 1 % normal serum from the same species as the Alexa Fluor®-FluoroNanogold^TM reagent, for 10 minutes to 1 hour at room temperature.
6. Rinse with PBS (3 X 1 min).
7. Postfix with 1 % glutaraldehyde in PBS at room temperature (3 mins).
8. Rinse in deionized water for (2 X 5 min).
9. If desired, contrast sections with uranyl acetate and/or lead citrate before examination.

PBS-Milk Buffer: 20 mM phosphate 150 mM NaCl pH 7.40 5% Non-fat dried milk (final concentration) Optional, may reduce background: 0.5 M NaCl 0.05% Tween 20 0.1% gelatin (high purity)

PBS Buffer: 20 mM phosphate 150 mM NaCl pH 7.40

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Special Considerations for Direct Viewing of Alexa Fluor®-FluoroNanogold^TM in the Electron Microscope

Alexa Fluor®-FluoroNanogold^TM provides a much improved resolution and smaller probe size over other colloidal gold antibody products. However, because NANOGOLD® is only 1.4 nm in diameter, it will not only be smaller, but will appear less intense than, for example, a 5 nm gold particle. With careful work, however, Alexa Fluor®-FluoroNanogold^TM may be seen directly through the binoculars of a standard EM even in 80 nm thin sections. However, achieving the high resolution necessary for this work may require new demands on your equipment and technique. Several suggestions follow:

1. Before you start a project with Alexa Fluor®-FluoroNanogold^TM it is helpful to see it so you know what to look for. Dilute the Alexa Fluor®-FluoroNanogold^TM stock 1:5 and apply 4 ml to a grid for 1 minute. Wick the drop and wash with deionized water 4 times.

2. View Alexa Fluor®-FluoroNanogold^TM at 100,000 X magnification with 10 X binoculars for a final magnification of 1,000,000 X. Turn the emission up full and adjust the condenser for maximum illumination.

3. The alignment of the microscope should be in order to give 0.3 nm resolution. Although the scope should be well aligned, you may be able to skip this step if you do step 4

4. Objective stigmators must be optimally set at 100,000 X. Even if the rest of the microscope optics are not perfectly aligned, adjustment of the objective stigmators may compensate and give the required resolution. You may want to follow your local protocol for this alignment but since it is important, a brief protocol is given here:
   1. At 100,000 X (1 X 10⁶ with binoculars), over focus, under focus, then set the objective lens to in focus. This is where there is the least amount of detail seen.
   2. Adjust each objective stigmator to give the least amount of detail in the image.
   3. Repeat steps a and b until the in focus image contains virtually no contrast, no wormy details, and gives a flat featureless image.

5. Now underfocus slightly, move to a fresh area, and you should see small black dots of 1.4 nm size. This is the NANOGOLD®. For the 1:5 dilution suggested, there should be about 5 to 10 gold spots on the small viewing screen used with the binoculars. Contrast and visibility of the gold clusters is best at 0.2 - 0.5 microns defocus, and is much worse at typical defocus values of 1.5 - 2.0 microns commonly used for protein molecular imaging.

6. In order to operate at high magnification with high beam current, thin carbon film over fenestrated holey film is recommended. Alternatively, thin carbon or 0.2% Formvar over a 1000 mesh grid is acceptable. Many plastic supports are unstable under these conditions of high magnification/high beam current and carbon is therefore preferred. Contrast is best using thinner films and thinner sections.

7. Once you have seen NANOGOLD® you may now be able to reduce the beam current and obtain better images on film. For direct viewing with the binoculars reduction in magnification from 1,000,000 X to 50,000 X makes the NANOGOLD® much more difficult to observe and not all of the golds are discernable. At 30,000 X (300,000 X with 10 X binoculars) NANOGOLD® particles are not visible. It is recommended to view at 1,000,000 X, with maximum beam current, align the objective stigmators, and then move to a fresh area, reduce the beam, and record on film.

8. If the demands of high resolution are too taxing or your sample has an interfering stain, a very good result may be obtained using silver enhancement to give particles easily seen at lower magnification.

Silver Enhancement of Alexa Fluor®-FluoroNanogold^TM for EM

Our LI SILVER silver enhancement system is convenient and not light sensitive, and suitable for all applications. Improved results in the EM may be obtained using HQ SILVER, which is formulated to give slower, more controllable particle growth and uniform particle size distribution.¹⁰

Specimens must be thoroughly rinsed with deionized water before silver enhancement reagents are applied. This is because the buffers used for antibody incubations and washes contain chloride ions and other anions which form insoluble precipitates with silver. These are often light-sensitive and will give non-specific staining. To prepare the developer, mix equal amounts of the enhancer and initiator immediately before use. Alexa Fluor®-FluoroNanogold^TM will nucleate silver deposition resulting in a dense particle 2-20 nm in size or larger depending on development time. Use of nickel grids is sometimes preferred.

Fluorescence microscopy should be performed BEFORE silver enhancement. This is because the fate of the fluorophores during silver enhancement has not been determined; deposition of silver may obscure the fluorescence.

The relevent procedure for immunolabeling should be followed. Silver enhancement is then performed as follows:

1. Rinse with deionized water (2 X 5 mins).
2. OPTIONAL (may reduce background): Rinse with 0.02 M sodium citrate buffer, pH 7.0 (3 X 5 mins).
3. Float grid with specimen on freshly mixed developer for 1-8 minutes, or as directed in the instructions for the silver reagent. More or less time can be used to control particle size. A series of different development times should be tried, to find the optimum time for your experiment. With HQ silver, a development time of 4 min. gives 15-40 nm round particles.
4. Rinse with deionized water (3 X 1 min).
5. Mount and stain as usual.

NOTE: Treatment with osmium tetroxide followed by uranyl acetate staining can lead to loss of the silver enhanced NANOGOLD® particles. This may be prevented by gold toning:¹¹

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.

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Gold Enhancement of Alexa Fluor®-FluoroNanogold^TM for EM

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Immunoblotting

1. Spot 1 microliter dilutions of the antigen in buffer 4 onto hydrated nitrocellulose membrane. Use an antigen concentration range from 100 ng to 0.01 pg / microliter.
2. Block with buffer 1 for 30 minutes at 37°C.
3. Incubate with primary antibody according to usual procedure (usually 1 or 2 hours).
4. Rinse with buffer 1 (3 X 10 mins).
5. Incubate with a 1/100 to 1/200 dilution of the Alexa Fluor®-FluoroNanogold^TM reagent in buffer 2 for 2 hours at room temperature.
6. Rinse with buffer 3 (3 X 5 mins), then buffer 4 (2 X 5 mins).
7. OPTIONAL (may improve sensitivity): Postfix with glutaraldehyde, 1 % in buffer 4 (10 mins).
8. Rinse with deionized water (2 X 5 mins).
9. OPTIONAL (may reduce background): Rinse with 0.05 M EDTA at pH 4.5 (5 mins).
10. Develop with freshly mixed silver developer for 20-25 minutes or as directed in the instructions for the silver reagent, twice. Rinse thoroughly with deionized water between developments to remove all the reagent.
11. Rinse several times with deionized water.

Other procedures may be used; for example the Alexa Fluor®-FluoroNanogold^TM reagent may be used as a tertiary labeled antibody, or a custom Alexa Fluor®-FluoroNanogold^TM conjugate may be the primary antibody. If additional antibody incubation steps are used, rinse with buffer 3 (3 X 10 mins) after incubation.

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References

1. "Combined ALEXA-488 and Nanogold Antibody Probes;" Hainfeld, J. F.; Furuya, F. R.; Powell, R. D., and Liu, W.; Microsc. Microanal., 8, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2002); Voekl, E.; Piston, D.; Gauvin, R.; Lockley, A. J.; Bailey, G. W., and McKernan, S., Eds.; Cambridge University Press, New York, NY, 2002, p. 1030CD (http://www.nanoprobes.com/MSAFN02.html).

2. Powell, R. D.; Hainfeld, J. F.; Halsey, C. M. R.; Joshi, V. N.; Hacker, G. W.; Hauser-Kronberger, C., and Takvorian, P. M.; Microsc. Microanal., 5, (Suppl. 2: Proceedings); G. W. Bailey, W. G. Jerome, S. McKernan, J. F. Mansfield, and R. L. Price (Eds.); Springer-Verlag, New York, NY; 1999, 478-479 (http://www.nanoprobes.com/MSAFN99.html); Powell, R. D.; Halsey, C. M. R., and Hainfeld, J. F.; Micros. Res. Technique, 42, 2-12 (1998); Powell, R. D., Halsey, C. M. R., Spector, D. L., Kaurin, S. L., McCann J., and Hainfeld, J. F.: J. Histochem. Cytochem., 45, 947-956 (1997).

3. Spector, D. L., and Smith, H. C.; Exp. Cell. Res., 163, 87 (1986).

4. Hainfeld, J. F., and Powell, R. D., Cell Vision, 4 308-324 (1997); Furuya, F. R., and Hainfeld, J. F.; J. Histochem. Cytochem., 40, 177 (1992); Furuya, F. R., Hainfeld, J. F., and Powell, R. D.; Proc. 49th Ann. Mtg., Electr. Micros. Soc. Amer.; G. W. Bailey and Hall, E. L., (Eds.); San Francisco Press, San Francisco, CA, 1991, p. 286 (http://www.nanoprobes.com/MSA92ng.html).

5. Tracz, E., Dickson, D. W., Hainfeld, J. F., and Ksiezak-Reding, H. Brain Res., 773, 33-44 (1997); Gregori, L., Hainfeld, J. F., Simon, M. N., and Goldgaber, D. Binding of amyloid beta protein to the 20S proteasome. J. Biol. Chem., 272, 58-62 (1997); Hainfeld, J. F.; Safer, D.; Wall, J. S.; Simon, M. N.; Lin, B. J., and Powell, R. D.; Proc. 52nd Ann. Mtg., Micros. Soc. Amer.; G. W. Bailey and Garratt-Reed, A. J., (Eds.); San Francisco Press, San Francisco, CA, 1994, p. 132 (http://www.nanoprobes.com/MSANV.html).

6. Hainfeld, J. F., and Furuya, F. R.; in Immunogold-Silver Staining: Principles, Methods and Applications (M. A. Hayat, Ed.), CRC Press, Boca raton, FL., 1995: pp. 71-96.

7. Krenács, T., and Krenács, L.; in Immunogold-Silver Staining: Principles, Methods and Applications (M. A. Hayat, Ed.), CRC Press, Boca raton, FL., 1995: pp. 57-69.

8. J. E. Beesley, in "Colloidal Gold: Principles, Methods and Applications," M. A. Hayat, (Ed.), Academic Press, New York, 1989; Vol. 1, pp. 421-425.

9. Lujan, R.; Nusser, Z.; Roberts, J. D. B.; Shigemoto R.; Ohishi, H., and Somogyi, P.: J. Chem. Neuroanat., 13, 219-241 (1997).

10. Humbel, B. M.; Sibon, O. C. M.; Stierhof, Y.-D., and Schwarz, H.: Ultra-small gold particles and silver enhancement as a detection system in immunolabeling and In Situ hybridization experiments; J. Histochem. Cytochem., 43, 735-737 (1995).

11. Arai, R.; Geffard, M.; and Calas, A. Intensification of labeling of the immunogold silver staining method by gold toning. Brain Res. Bull. 28, 343-345 (1992).

12. Moeremans, M.; Daneels, G.; Van Dijck, A.; Langanger, G, and De Mey, J. Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assays with immunogold and immunogold/silver staining. J. Immunol. Meth. 74, 353 (1984).

13. Robinson, J. M.; Takizawa, T.; Pombo, A., and Cook, P. R. Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap. J. Histochem. Cytochem., 49, 803-8 (2001).

14. Robinson, J. M.; Takizawa, T., and Vandre, D. D. Applications of gold cluster compounds in immunocytochemistry and correlative microscopy: comparison with colloidal gold. J. Microsc., 199, 163-79 (2000).

15. Takizawa, T., and Robinson, J. M. FluoroNanogold^TM is a bifunctional immunoprobe for correlative fluorescence and electron microscopy. J. Histochem. Cytochem., 48, 481-6 (2000).

16. Takizawa, T., and Robinson, J. M. Analysis of antiphotobleaching reagents for use with FluoroNanogold^TM in correlative microscopy. J. Histochem. Cytochem., 48, 433-6 (2000).

17. Takizawa, T., Suzuki, K., and Robinson, J. M. Correlative microscopy using FluoroNanogold^TM on ultrathin cryosections. Proof of principle. J. Histochem. Cytochem., 46, 1097-102 (1998).

For a complete list of references citing this product, please visit our References page.

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