FAB-3 PRODUCT INFORMATION

FAB-3 Gold-Antibody Conjugates
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Contents

• Product Information
• General Considerations for Immunostaining with FAB-3 Reagents
• Thiol Caution
• Electron Microscopy Immunolabeling with FAB-3
  1. Cells in Suspension
  2. Thin Sections
• Silver Enhancement of FAB-3 for EM
• Immunolabeling and Silver Enhancement with FAB-3 for Light Microscopy
• Immunoblotting
• 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

Contents

General Considerations for Immunostaining with FAB-3 Reagents

Note: some variation in diameter is inherent in gold particles of this size.

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Thiol Caution

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Electron Microscopy Immunolabeling with FAB-3

Cells in Suspension

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-BSA as described in step 1 (2 X 5 mins). Resuspend in 1 ml Buffer 1.
5. Dilute FAB-3 ~ 50 times in PBS-BSA buffer and add 30 microliters to cells; incubate for 30 minutes with occasional shaking.
6. Wash cells in PBS-BSA 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 buffer 1 (3 X 5 mins).

PBS-BSA Buffer:
20 mM phosphate with 150 mM NaCl, pH 7.40
0.5% BSA
0.1% gelatin (high purity)
optional, may reduce background:
0.5 M NaCl
0.05% Tween 20

PBS Buffer:
20 mM phosphate with 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 (Ref. 2) and NANO-W negative stains are recommended for use with FAB-3 reagents for highest contrast imaging.

Thin Sections

Labeling with FAB-3 may be performed before or after embedding (Ref. 3). Labeling before embedding and sectioning (the pre-embedding method) is used for the study of surface antigens, particularly small organisms such as viruses budding from host cells. It gives good preservation of cellular structure, and subsequent staining usually produces high contrast for study of the cellular details. Labeling after embedding and sectioning (the post-embedding method) allows the antibody access to the interior of the cells, and is used to label both exterior and interior features. 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. All commonly used embedding media may be used in FAB-3 labeling experiments.

PROCEDURE FOR PRE-EMBEDDING METHOD (Ref. 3):

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-BSA (30 mins - 1 hour, or usual time. Buffer formulations are given below.
4. Rinse with PBS-BSA (3 X 1 min).
5. Incubate with FAB-3 reagent diluted 1/40 - 1/200 in PBS-BSA with 1 % normal serum from the same species as the FAB-3 reagent, for 10 minutes to 1 hour at room temperature.
6. Rinse with PBS-BSA (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. Dehydrate and embed according to usual procedure.
10. Stain (uranyl acetate, lead citrate or other positive staining reagent) as usual before examination.

Silver enhancement may be performed before or after embedding (see below); it should be completed before postfixing or staining with osmium tetroxide, uranyl acetate or similar reagents is carried out.

PROCEDURE FOR POST-EMBEDDING METHOD (Ref. 3):

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-BSA (1 hour or usual time. Buffer formulations are given below.
4. Rinse with PBS-BSA (3 X 1 min).
5. Incubate with FAB-3 reagent diluted 1/40 - 1/200 in PBS-BSA with 1 % normal serum from the same species as the FAB-3 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.

Silver enhancement may also be used to enlarge the FAB-3 gold particles (see below). Silver enhancement should be completed before stains such as uranyl acetate, osmium tetroxide or lead citrate are applied.

PBS-BSA Buffer:
20 mM phosphate with 150 mM NaCl, pH 7.40
0.5% BSA
0.1% gelatin (high purity)
optional, may reduce background:
0.5 M NaCl
0.05% Tween 20

PBS Buffer:
20 mM phosphate with 150 mM NaCl, pH 7.40

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Silver Enhancement of FAB-3 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 more controllable particle growth and uniform particle size distribution, at neutral pH for best possible ultrastructural preservation.

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. FAB-3 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).

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. 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.
3. Rinse with deionized water (3 X 1 min).
4. Mount and stain as usual.

Fixing with osmium tetroxide may cause some loss of silver; if this is found to be a problem, slightly longer development times may be appropriate.

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Immunolabeling and Silver Enhancement with FAB-3 for Light Microscopy

Samples must be rinsed with deionized water before silver enhancement. This is because the reagent contains silver ions in solution, which react to form a precipitate with chloride, phosphate and other anions which are components of buffer solutions. The procedure for immunolabeling with FAB-3 and silver enhancement is given below.

1. Spin cells onto slides using Cytospin, or use parafin section.
2. Incubate with 1 % solution of bovine serum albumin in PBS (PBS-BSA) for 10 minutes to block non-specific protein binding sites.
3. Incubate with primary antibody, diluted at usual working concentration in PBS-BSA (1 hour or usual time)
4. Rinse with PBS-BSA (3 X 2 min).
5. Incubate with FAB-3 reagent diluted 1/40 - 1/200 in PBS-BSA with 1 % normal serum from the same species as the FAB-3 reagent, for 1 hour at room temperature.
6. Rinse with PBS (3 X 5 min).
7. Postfix with 1 % glutaraldehyde in PBS at room temperature (3 mins).
8. Rinse with deionized water (3 X 1 min).
9. Develop specimen with freshly mixed developer for 5-20 minutes, or as directed in the instructions for the silver reagent. More or less time can be used to control intensity of signal. A series of different development times may be used, to find the optimum enhancement for your experiment; generally a shorter antibody incubation time will require a longer silver development time.
10. Rinse with deionized water (2 X 5 mins).
11. The specimen may now be stained if desired before examination, with usual reagents.

PBS-BSA Buffer:
20 mM phosphate with 150 mM NaCl, pH 7.40
0.5% BSA
0.1% gelatin (high purity)
optional, may reduce background:
0.5 M NaCl
0.05% Tween 20

PBS Buffer:
20 mM phosphate with 150 mM NaCl, pH 7.40

To obtain an especially dark silver signal, the silver enhancement may be repeated with a freshly mixed portion of developer.

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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 FAB-3 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.

CAUTION: FAB-3 particles degrade upon exposure to concentrated thiols such as beta-mercaptoethanol or dithiothreitol. If such reagents must be used, concentrations should be kept below 1 mM and exposure restricted to 10 minutes or less.

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
0.8% BSA
1% normal serum; use serum of the host animal for the FAB-3 antibody
0.1% gelatin (Type B, approx. 60 bloom)
optional, may reduce background:
0.5 M NaCl
0.05% Tween 20

Buffer 3: 20 mM phosphate, 150 mM NaCl, pH 7.40
0.8% BSA (bovine serum albumin)
2 mM sodium azide

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

Other procedures may be used; for example the FAB-3 reagent may be used as a tertiary labeled antibody, or a custom FAB-3 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. Baschong, W., and Wrigley, N. G.; Journal of Electron Microscopy Technique, 14, 313 (1990).
2. 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.
3. J. E. Beesley, in "Colloidal Gold: Principles, Methods and Applications," M. A. Hayat, ed., Academic Press, New York, 1989; Vol. 1, pp. 421-425.
4. Moeremans, M. et al., J. Immunol. Meth.; 74, 353 (1984).

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