FLUORONANOGOLD* PRODUCT INFORMATION

Product Name:	FLUORONANOGOLD-Streptavidin conjugate
Catalog Number:	7016
Appearance:	Fluorescent pale greenish-yellow solution
Revision:	1.6 (April 2003)

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  Material Safety Data Sheet (PDF)

Contents

1. Product Information
2. General Considerations for Immunostaining with FLUORONANOGOLD Reagents
3. Using Stains with FLUORONANOGOLD
4. Thiol Caution
5. Temperature Caution
6. In Situ Hybridization with FLUORONANOGOLD®-Streptavidin
7. Fluorescence Microscopy with FLUORONANOGOLD
8. Electron Microscopy Immunolabeling with FLUORONANOGOLD
   1. Cells in Suspension
   2. Thin Sections
9. Special Considerations for Viewing FLUORONANOGOLD in the Electron Microscope
10. Silver Enhancement of FLUORONANOGOLD for EM
11. Gold Enhancement of FLUORONANOGOLD for EM
12. Immunoblotting
13. 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.

* Patented technology.

Product Information

Figure 1: Streptavidin conjugated to Fluorescein and NANOGOLD® via primary amines to give FLUORONANOGOLD.

Contents

General Considerations for Immunostaining with FluoroNanogold Reagents

Properties

• FLUORONANOGOLD contains an extremely uniform 1.4 nm diameter gold particle (ca.10%).

• Streptavidin-FLUORONANOGOLD is smaller than a single whole IgG molecule. It is not significantly larger than Streptavidin-NANOGOLD®, the smallest gold immunoprobe commercially available, and will penetrate and reach antigens inaccessible to other gold probes.

• Streptavidin - FLUORONANOGOLD 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 FLUORONANOGOLD label to 1 streptavidin make this product distinct from the 0.2 - 10 variable stoichiometry of other colloidal gold - protein preparations.

• FLUORONANOGOLD particles do not have affinity to proteins as do colloidal golds. This reduces background and false labeling.

• FLUORONANOGOLD 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 Stains with FluoroNanogold

1. Use of reduced amounts or concentrations of usual stains.
2. Use of lower atomic number stains such as NANOVAN, a Vanadium based stain.⁴
3. Enhancement of FLUORONANOGOLD with silver developers, such as LI SILVER or HQ SILVER.
4. Enhancement of FLUORONANOGOLD with the gold developer, GoldEnhance.

Thiol Caution

Temperature caution

Contents

Methods

Contents

In Situ Hybridization with FLUORONANOGOLD-Streptavidin

Procedure (adapted from Hacker, G.W. et al)^7,8):

Practical considerations: This is a robust and reliable technique for routine use. It is intended for biotinylated hybridization probes. Other types of reporter molecules may be demonstrated by application of a biotinylated linking antibody system. The sensitivity of Nanogold-silver ISH depends, to a large degree, at the dilution of streptavidin-Nanogold and the duration of silver development applied. Careful adjustment of the protease predigestion is necessary. In some preparations, some degree of unwanted background staining in connective tissue is obtained. This is in part due to fixation and possible excessive protease treatment and can often be reduced by application of higher dilutions of streptavidin-Nanogold.

Fluorescence microscopy should be performed before silver enhancement since the silver deposition process results in removal of fluorescence.

1. Deparaffinize sections from formaldehyde-fixed tissue in fresh xylene (2 times 15 min each).
2. Rinse and rehydrate in graded alcohols and distilled water (2-3 min each).
3. Soak in phosphate-buffered saline (PBS, 20mM, pH 7.6) for 3 min.
4. Incubate with 0.1 mg/mL proteinase K (code no. 1 373 196, from Boehringer Mannheim, Mannheim, FRG) in PBS at 37°C for about 8 min. The duration is critical and has to be tested very carefully, depending on tissue, fixation and other factors.
5. Rinse in 2 changes of PBS, 3 min.
6. Permeabilize with 0.3% Triton X-100 in PBS for 15 min.
7. Wash in PBS for 2 min.
8. Rinse in 2 changes of distilled water, dehydrate with graded alcohols (50%, 70%, 98% isopropanol) for 1 min each and air-dry the sections.
9. Prehybridize with 1:1 mixture of deionized formamide and 20% dextran sulfate in 2X SSC at 50°C for 5 min.
10. Carefully shake off excess prehybridization block.
11. Add one drop of biotinylated DNA probe on the section and cover with a small coverslip. Avoid air bubbles.
12. Heat sections on heating block at 92-94°C for 8-10 min to denature DNA.
13. Incubate in a moist chamber at 37°C overnight (or for at least 2 hours).
14. Post-hybridization washes (5 min each): 2 changes of 4X SSC (1st wash to remove coverslips), 2X SSC, 0.1X SSC, 0.05X SSC, and then distilled water.
15. Put slides into Lugol's iodine solution (Merck, Darmstadt, Germany) for 5 min.
16. Wash in tap water and then distilled water.
17. Put into 2.5% sodium thiosulfate for a few seconds until sections are colorless. Then wash in tap water for 5 min and distilled water for 2 min.
18. Immerse in PBS containing 0.1% fish gelatin (45% concentrate - Cat. No. G-7765, Sigma-Aldrich, Steinheim, Germany) and 0.1% Tween-20 for 5 min.
19. Incubate sections with FluoroNanogold-streptavidin diluted 1:200 to 1:500 in PBS containing 1% BSA at room temperature for 60 min.
20. Wash in 3 changes of PBS containing 0.1% fish gelatin and 0.1% Tween-20 for 5 min each.
21. Repeatedly wash in distilled water for at least 10 min altogether, the last 2 rinses in ultrapure water (EM-grade).

    The specimen may now be observed by fluorescence microscopy.

22. Perform silver acetate autometallography or GoldEnhance development (Nanoprobes, see Protocol 4).
23. Rinse carefully in tap water for at least 3 min. After silver amplification, sections can be counterstained with Nuclear Fast Red, dehydrated and mounted in Permount or in DPX (BDH Chemicals, Poole, UK). Do not use Eukitt.

Solutions:

Phosphate-buffered saline (PBS): 10X PBS (Mg²⁺ and Ca²⁺-free) pH 7.6: 11.36 g Na₂HPO₄, 2.72 g KH₂PO₄, 87.0 g NaCl in 800 mL distilled water. Adjust pH with concentrated NaOH and add distilled water to a final volume of 1 L.

Standard Sodium Citrate Buffer (SSC): 175.32 g NaCl and 88.23 g sodium citrate in 800 ml distilled water. Adjust pH with NaOH to 7.0 and add distilled water to a final volume of 1 L.

Silver Development:

1. Silver amplification: Place the slides vertically in a glass container (preferably with about 80 mL volume and up to 19 slides; Schiefferdecker-type) and cover them with the mixture of solutions A and B. Staining intensity can be checked in the light microscope during the amplification process, which usually takes about 5-20 min, depending on primary antibody or nucleic acid probe concentration, incubation conditions, and the amount of accessible antigen or nucleic acid sequence in question.
2. Stop enhancement by washing in distilled water (several changes).
3. After stopping the enhancement process, slides can be examined in a light microscope more carefully. If staining intensity is still too low, wash slides for one more time in double-distilled water and develop further in enhancement solution.

Reagents (Solutions A and B should be freshly prepared for every run):

Solution A: Dissolve 80 mg silver acetate (code 85140; Fluka, Buchs, Switzerland) in 40 mL of glass double-distilled water. (Silver acetate crystals can be dissolved by continuous stirring within about 15 min.)

Citrate buffer: Dissolve 23.5 g of trisodium citrate dihydrate and 25.5 g citric acid monohydrate in 850 mL or deionized or distilled water. This buffer can be kept at 4°C for at least 2-3 weeks. Before use, adjust to pH 3.8 with citric acid solution.

Solution B: Dissolve 200 mg hydroquinone in 40 mL citrate buffer.

Enhancement solution: Just before use, mix solution A with solution B.

Alternative autometallography procedure (using LI Silver, Nanoprobes):

1. Allow LI Silver to come to room temperature.
2. Mix equal amounts of A and B solutions of LI Silver (red and blue capped bottles).
3. Immediately cover sections with developer.
4. Keep in subdued light, but check sections occasionally, using eye or light microscope. Development usually takes 20 min to 45 min.
5. Stop development by rinsing in deionized water. Alternatively, a fixer may be used to stop development immediately. Use a photographic fixer (e.g., Agefix, Agfa Gevaert, FRG, diluted 1:20) (can be reused). Treat for ~1 min. Another alternative is a 2.5% aqueous solution of sodium thiosulfate.
6. If sections are overdeveloped, they may be "back-developed" by 0.2 % Farmer's solution (9 parts sodium thiosulfate + 1 part potassium ferricyanide; form by mixing 0.18 g Na₂S₂O₃. 5H₂O + 0.02 g K₃Fe(CN)₆ in 100 ml water). Reversal may be halted with water.
7. Rinse slides carefully in tap water for at least 3 min. After silver amplification, sections can be counterstained with hematoxylin and eosin or nuclear fast red, dehydrated, and mounted in DPX (BDH Chemicals, UK).

Contents

Fluorescence Microscopy Immunolabeling with FluoroNanogold

The procedure below² describes an example of the use of a FLUORONANOGOLD conjugate as a secondary probe. Dilutions of FluoroNanogold will vary with different procedures, but a 5-fold or 10-fold dilution is advisable as a starting point for most applications. Other protocols and techniques used with fluorescently-labeled antibodies may also be used with FLUORONANOGOLD. 10 mM biotin should be added to the third wash in step 8; this is because vacant biotin binding sites can quench the fluorescence of the FLUORONANOGOLD label, and should therefore be blocked before observation. It should also be noted that the fluorescence intensity of fluorescein is pH-dependent: it is maximized at pH 9.0 or higher, reducing to approximately 85 % at pH 7 and decreasing rapidly at lower pH values. Therefore, we recommend that the buffer used for the final wash should have a pH value of 7.4 or higher.

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. Permaeabilize in 0.2 % Triton X-100 plus 1 % normal goat serum (NGS) 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 biotinylated 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. Alternatively, a tertiary labeling procedure may be used where the primary antibody is not biotinylated, but the second antibody is. If additional antibody incubation steps are used, rinse with PBS with 1 % NS (3 X 10 mins) after incubation.
6. Wash in PBS with 1% NS + 5% non-fat dried milk (3 X 10 mins).
7. Incubate with FLUORONANOGOLD-Streptavidin 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.

Contents

Electron Microscopy with FluoroNanogold-Streptavidin

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.
2. Centrifuge cells (e.g. 1 ml at 10⁷ cells/ml) at 300 X g, 5 minutes; discard supernatant; resuspend in 1 ml buffer. Repeat this washing (centrifugation and resuspension) 2 times.
3. Incubate cells with 0.02 M glycine in PBS (5 mins). Centrifuge, then resuspend cells in PBS-milk buffer (specified below) or PBS containing 1 % BSA for 5 minutes.
4. Place 50 - 200 microliters of cells into Eppendorf tube and add 5 - 10 microliters of biotinylated primary antibody. Incubate 30 minutes with occasional shaking (do not create bubbles which will denature proteins). Alternatively, a tertiary labeling procedure may be used where the primary antibody is not biotinylated, but the second antibody is. If additional antibody incubation steps are used, rinse with PBS-milk buffer (3 X 10 mins) after incubation.
5. Wash cells using PBS-Milk as described in step 2 (2 X 5 mins). Resuspend in 1 ml PBS-Milk buffer.
6. Dilute FLUORONANOGOLD ~ 5 to 10 times in PBS-Milk buffer and add 30 microliters to cells; incubate for 30 minutes with occasional shaking.
7. Wash cells in PBS-BSA buffer as described in step 1 (2 X 5 mins).
8. 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).

0.1% gelatin (high purity)

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

PBS Buffer: 20 mM phosphate 150 mM NaCl pH 7.40

Negative staining 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 negative stain is specially formulated for use with NANOGOLD® reagents;⁴ it is based on vanadium, which gives a lighter stain than uranium, lead or tungsten-based negative stains and allows easier visualization of FLUORONANOGOLD particles with little or no silver enhancement.

Other procedures may be used; for example the NANOGOLD® reagent may be used as a tertiary labeled antibody in a system where a biotinylated secondary antibody is used with an unlabeled primary antibody. If additional antibody incubation steps are used, rinse with PBS-BSA (3 X 5 mins) after incubation.

Thin Sections

Labeling with FluoroNanogold™-Streptavidin may be performed before (the pre-embedding method)^9,10 or after embedding and sectioning (the post-embedding method).^9,10 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:⁹

If specimen has already been labeled with FLUORONANOGOLD, 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 biotinylated 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). Alternatively, a tertiary labeling procedure may be used where the primary antibody is not biotinylated, but the second antibody is. If additional antibody incubation steps are used, rinse with buffer 3 (3 X 10 mins) after incubation.
4. Rinse with PBS-Milk (3 X 1 min).
5. Incubate with FLUORONANOGOLD reagent diluted 1/5 - 1/20 in PBS- Milk with 1 % normal serum 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:⁹

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 biotinylated 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). Alternatively, a tertiary labeling procedure may be used where the primary antibody is not biotinylated, but the second antibody is. If additional antibody incubation steps are used, rinse with buffer 3 (3 X 10 mins) after incubation.
4. Rinse with PBS-Milk (3 X 1 min).
5. Incubate with FLUORONANOGOLD reagent diluted 1/5 - 1/20 in PBS- Milk with 1 % normal serum, 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.

0.1% gelatin (high purity)

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

PBS Buffer: 20 mM phosphate 150 mM NaCl pH 7.40

Contents

Special Considerations for Direct Viewing of FluoroNanogold in the Electron Microscope

FLUORONANOGOLD provides a much improved resolution and smaller probe size over other colloidal gold antibody products. However, because FLUORONANOGOLD 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, FLUORONANOGOLD 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 FLUORONANOGOLD it is helpful to see it so you know what to look for. Dilute the FLUORONANOGOLD 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 FLUORONANOGOLD 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 FLUORONANOGOLD. 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 m defocus, and is much worse at typical defocus values of 1.5 - 2.0 m 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 FLUORONANOGOLD 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 FLUORONANOGOLD much more difficult to observe and not all of the golds are discernable. At 30,000 X (300,000 X with 10 X binoculars) FLUORONANOGOLD 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 FluoroNanogold-Streptavidin 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.¹¹

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. FLUORONANOGOLD will nucleate silver deposition resulting in a dense particle 2-20 nm in size or larger depending on development time. Use nickel grids (not copper).

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 up to step 7 as described above. 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 much more drastic loss of the silver enhanced NANOGOLD® particles. This may be prevented by gold toning:¹²

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.

Contents

Gold Enhancement of FluoroNanogold for EM

Contents

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 biotinylated primary antibody according to usual procedure (usually 1 or 2 hours). Alternatively, a tertiary labeling procedure may be used where the primary antibody is not biotinylated, but the second antibody is. If additional antibody incubation steps are used, rinse with buffer 3 (3 X 10 mins) after incubation.
4. Rinse with buffer 1 (3 X 10 mins).
5. Incubate with a 1/100 to 1/200 dilution of the FLUORONANOGOLD 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). Fluorescence may be observed at this point.
9. OPTIONAL (may reduce background): Rinse with 0.05 M EDTA at pH 4.5 (5 mins).
10. Develop with freshly mixed silver developer (LI SILVER) for 15 – 30 minutes, or GoldEnhance LM/Blot for 10-20 minutes (or as directed in the instructions for these reagents). Development may be repeated (develop, rinse, develop with freshly mixed developer), to reduce background. Rinse thoroughly with deionized water between developments to remove all the reagent.
11. Rinse several times with deionized water.

Buffer 1: 20 mM phosphate 150 mM NaCl pH 7.40 4% BSA (bovine serum albumin) 2 mM sodium azide (NaN3)

Buffer 2: 20 mM phosphate 150 mM NaCl pH 7.4 5% non-fat dried milk 1% normal serum; use serum of the host animal for the FLUORONANOGOLD® antibody. Optional, may reduce background: 0.1% gelatin (Type B, approx. 60 bloom) 0.5 M NaCl 0.05% Tween 20

Buffer 3: 20 mM phosphate 150 mM NaCl pH 7.40 5% non-fat dried milk 2 mM sodium azide

Buffer 4 (PBS): 20 mM phosphate 150 mM NaCl pH 7.4

Other procedures may be used; for example, FLUORONANOGOLD-Streptavidin may be used as a tertiary labeled probe for a biotinylated secondary antibody, or as a probe for a directly biotinylated target. If additional antibody incubation steps are used, rinse with buffer 3 (3 X 10 mins) after incubation.

Contents

References

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14. 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).

15. 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).

16. 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).

17. 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).

18. 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).

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