Ultra-small gold particles and silver enhancement as a detection system in immunolabeling and in situ hybridization experiments.

Reading recent articles published in the Journal of Histochemistry and Cytochemistry (5.6) we became aware that the advantages offered by ultrasmall gold probes as a detection system in immunolabeling and in situ hybridization experiments may still not be fully exploited. This letter summarizes the merits of ultra-small gold probes and gives some advice for their successful application. Here, gold particles (whether colloids or not) that are smaller than 1.5 nm (usually -1 nm) are called ultra-small gold particles. Ultra-small gold markers are available from several companies: AuroProbe One (Amenham; Poole, UK). GP-Ultra Small (Aurion; Wageningen. The Netherlands), 1nm gold (British BioCell; Cardiff, UK), and Nanogold (Nanoprobes; Stony Brook, NY). I t should be mentioned that some preparations also contain particles considerably larger than 1.5 nm (see Figure 3B in ref. 5) (18.19). The main advantage of ultra-small gold particles compared with larger ones is increased detection efficiency. First, ultra-small gold probes penetrate more easily. Gold probes of different sizes (ultra-small, 7 nm or 10 nm) can penetrate into HeLa cells permeabilized with Triton X-100 to reveal the intermediate filament vimentin. In the same sample, however, a component of the internal nuclear matrix could be visualized only with the ultra-small gold probe (7). Obviously, the dense meshwork of the lamina-pore complex and/or the highly negatively charged DNA prevent larger particles from obtaining access to intranuclear antigens. Second, labeling efficiency increases with decreasing diameter of the gold probe used (11). This increase is even more prominent for the ultra-small gold particles (5.9.12). In preand post-embedding in situ hybridization experiments, a comparable improvement of labeling efficiency using ultra-small gold particles was found (16.17). In summary, secondary antibodies tagged to ultra-small gold particles more efficiently detect primary antibodies than those tagged to larger gold particles. The increase in label efficiency using ultra-small gold probes is less pronounced if surfaces of resin sections (e.g. methacrylates such as Lowicryls and epoxy resins) are labeled. However. if the density of the matrix of the section is lower [e.g., thawed frozen sections (21) or cryostat sections]. the ultra-small gold particles have a better chance to penetrate. In conventional electron microscopy, the ultra-small gold particles are difficult to visualize, especially on stained ultra-thin sections (19.20). Thercfore, the size of the ultra-small gold particles must be increased by controlled silver deposition (autometallography). We extensively investigated four published protocols (1.3.4.8.13) along with four commercial developers (IntenSE M. Amersham; R-Gent. Aurion; SEKL 15, British BioCell; HQ Silver, Nanoprobe). In our hands, only the enhancers containing the socalled protective colloid gum arabic (3.14) gave good results with respect to evenness of enhancement, to regularity of shape of the final particles, and to the number of particles enhanced (which determines the efficiency of detection) (see 18-20 for review). Interestingly, less dficiently working enhancerscan be improved by adding gum arabic (18,W). We have not tested