Once the minimum amount of protein necessary to stabilize a given quantity of gold is known, any quantity of gold probe can be produced.
1. Dissolve the required amount of protein in 0.1-0.2 mL of distilled water in a centrifuge tube and add 10 mL of the gold sol.
2. After 2 min, add 1 mL of 1% aqueous polyethylene glycol solution to stabilize the gold probe (8).
3. Centrifuge the mixture at a speed depending on the size of the gold complex: 15 nm at 60,000g for 1 h at 4°C (8), 12 nm at 50,000g for 45 min at 4°C (4), 5-12 nm at 105,000g for 1.5 h at 4°C (8), 5 nm at 125,000g for 45 min at 4°C (5), and 2-3 nm at 105,000g for 1.5 h at 4°C (8). The pellet formed consists of two phases (8). There is a large loose part, which is the protein-gold complex. In addition, there is a tight, dense pellet on the side of the tube, which contains aggregated gold particles and gold particles that have not been fully stabilized.
4. Resuspend the loose part of the pellet in 1.5 mL of PBS containing 0.2 mg/mL of polyethylene glycol. This can be stored for up to 1 yr at 4°C. If necessary, 0.5 mg/mL of sodium azide may be added to prevent small organisms from growing in the probe. It should then be suitably diluted before use.
The previously described technique is the basis for the preparation of colloidal gold probes and the principles of probe production are identical for each type of probe. Those who are interested should refer to Slot and Geuze (5), for preparation of protein A-gold probes, Roth (8) for production of antibody-gold complexes, Tolsen et al. (11) for production of the avidin-gold complex, Horisberger (10) for production of the lectin-gold complex, and Bendayan (12) for production of the enzyme-gold complex. New applications for gold probes are regularly appearing in the literature (e.g., see Notes 13 and 14) and a range of websites are now available for keeping researchers abreast of latest technological advances (see Note 15).
1. There are many ways of testing the probe, but the most convincing is by using a known positive sample. Therefore, this could be a histological section, an electron micrograph specimen, or a dot-blot. Estimation of the concentration of the probe by optical density measurements is a good method to standardize the concentration of probes from one batch to another, but in addition, it is always preferable to test the performance of the probes on known positive samples.
2. It is reasonably easy to make good-quality probes in the laboratory, but the investigator has to consider nonscientific factors, such as time and cost. Many gold conjugates are available commercially, in particular the commonly used secondary antibodies used in indirect im-munocytochemistry. A good compromise is to purchase the reagents that will be in constant demand and make the required quantity of those that are not readily available. When purchasing probes, it is important to seek information on the properties of the probe. Commercial gold probes are usually sold with certain variables closely controlled. The degree of particle aggregation is stated and the optical density of the solution given as a guideline to the concentration of the probe. Particle size variance will have been calculated by one of two methods. A laser diffraction particle sizer (13) might have been used or, alternatively, electron micrographs may have been made from Formvar grid preparations of the gold sol, particle sizes being measured by image analysis of the micrograph. In addition, especially when using double or triple labeling in transmission electron microscopy, the distribution of gold particle sizes and the variance should be noted—a 5-nm probe may comprise a particle size range of 3-6 nm, of which a large number of particles may potentially overlap with those from 1-nm or 10-nm samples with wide distributions.
3. Use of metallic gold probes in electron microscopy: immunogold labeling is a well-established procedure in both transmission and scanning electron microscopy. The ability to bind a range of proteins to gold probes of different but well-defined size offers great scope for single, double or even triple labeling at the electron microscopy level. The different applications for gold probes in electron microscopy have been reviewed in detail (14).
4. The use of metallic gold probes in light microscopy: using larger (e.g., 30-nm) gold labels, it is sometimes possible to build-up enough gold label in frozen or paraffin sections to enable antigen localization at the light microscope level. This technique is no more sensitive, however, than immuno-enzyme methods. Gold particles as small as 1 ^m may be located by nanometer particle video microscopy (Nanovid; ref. 15), but the ideal way to increase the sensitivity of immunogold labeling is by silver enhancement (immunogold-sil-ver staining; ref. 16).
5. Specialist microscopy for viewing gold labeling. Because of the light reflecting properties of gold (17) and silver (18) particles, it is possible to use either darkground or epipolarization microscopy (19,20) for the enhanced visualization of both types of labeling at the light microscope level. The technique is especially useful when used in conjunction with transmitted ordinary light as other tissue structures may be examined at the same time. Epipolarization-bright field double illumination may also be used for image analysis of multilabeling immunocytochemistry (21). Immunogold visualization in highly sensitive capping experiments with leucocyte surface proteins has been demonstrated using confocal scanning microscopy (22). Such methodology has helped to bridge the gap between light and electron microscopical studies.
6. It has been reported that some commercial preparations of colloidal gold-antibody complexes may contain free active antibody. Such free antibody will compete with antibody-colloidal gold particles for antigen binding sites and may reduce labeling intensity. The presence of free protein may be identified using a simple test procedure (23).
7. The use of probes prepared by a covalent attachment procedure of gold particles and proteins has been recently described as offering a number of advantages over conventional gold probes, including better resolution, stability, uniformity, sensitivity and complete absence of aggregation (24).
8. Recent advances in colloidal gold technology: there continue to be new frontiers in gold labeling that merit exploration. These include ultrasmall colloidal gold probes, new metal clusters, clusters with novel binding functionalities, new metal cluster conjugates with other molecules, and fluorescent metal cluster probes.
9. Ultrasmall colloidal gold probes: enhanced labeling efficiency and possibly greater penetration into samples have been associated with small gold particle sizes (25-27). Colloidal gold particles in the size range of 1 to 3 nm have been generated for immunolabeling. The use of 2-3 nm "thiocyanate-gold" probes was first described in 1986 and refinements of guidelines for preparing ultrasmall gold particles published in 1998 (28,29). Despite the potential advantages associated with enhanced labeling and penetration, ultrasmall probes have in practice been used less frequently than larger colloidal gold particles (>5 nm) mainly because the smaller particles require sophisticated imaging equipment for detection in routine cell or tissue preparations. Ultrasmall colloidal gold has several disadvantages, namely, the size range of the particles is very large and aggregation with antibodies leads to large impenetrable particles, and probe attachment is less stable.
10. Gold Clusters: an alternative approach to colloidal gold labeling of immunoprobes has been the development and use of metal cluster compounds (e.g., gold clusters; refs. 30-32) that are in the 0.8-1.4 nm range. Gold clusters have a core of multiple gold atoms. Undecagold has a core of 11 gold atoms with a diameter of 0.82 nm (33,34). The cluster is made water-soluble by altering covalently attached organic groups on the surface of the cluster, followed by linking to various proteins. A larger gold cluster is the 1.4-nm nanogold particle, which contains in the region of 67 gold atoms in its core and is covalently linkable to proteins (35). Silver enhancement is often needed to make the probes more visible, the smallest being undetectable even under electron microscopy. Indeed, the rationale behind many applications involves labeling with the tiny, highly penetrative clusters and then employing silver enhancement for visualization. However, enhancement is variable so that quantitative electron microscopy work is unreliable. Selected deposition of atomic gold onto the probes, instead of silver, has been suggested as a more controllable method of enhancement (36). If osmium tetroxide post fixation is required after silver enhancement, the silver shell should be stabilized by gold chloride toning (37,38).
11. Greengold: it is a chromatographically distinct component of Nanogold having a green color which, by gel filtration, runs ahead of a the similar-sized (1.4 nm) brown cluster; greengold accounts for about 50% of a nanoprobe preparation. Greengold is a highly stable, heavy atom label that has found to be most generally useful in STEM. The particle size, uniformity, scattering properties, and S/N ratio of these clusters was described by Wall in 1999 (39).
12. Fluoronanoprobes: antibody (whole immunoglobin G or fragment antigen binding fragment) can be conjugated to the 1.4-nm cluster nanogold and a fluorochrome to produce a probe that permits the correlative microscopical observation of the same cell profiles labeled in a single procedure using two imaging techniques (40). For example, high-resolution correlation between fluorescence microscopy and electron microscopy images may be performed in the same ultrathin sections and the procedure is especially useful when employed with antiphotobleaching methods (41-43).
13. Gold-facilitated autometallographic in situ hybridization (GOLDFISH) has been introduced as a bright-field alternative to established FISH for direct visualization of gene amplification using conventional microscopy (44,45). Streptavidin-nanogold is used to generate bright-field gene copy signals using gold-based autometallography, catalyzed reported deposition, and a biotin-labeled probe.
14. Gold labeling may be used in the recently introduced catalyzed reporter deposition-immunogold technique where biotinylated tyramide molecules are attached the antibody-antigen complex site; the biotinylated sites are visualized by interaction with streptavidin-gold (46,47).
15. Various websites provide extensive information on gold probe preparation and use. Of particular value are the regular Nanoprobe newsletters (http://www.nanoprobes.com) and immunogold newsletters (http://www.british-biocell.co.uk).
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