CRC - Gold and Silver Staining: Techniques in Molecular Morphology
|Publication Date:||17 April 2002|
The search for highly sensitive in situ antigen and nucleotide detecting techniques recently achieved a new milestone with the microscopic detection of single molecules.
In 1981 and 1983, two genius scientists, Gorm Danscher (Denmark) and Clive Holgate (U.K.), successfully developed silver enhancement (autometallography [AMG]) and immunogold-silver staining. The latter method uses the very small particles of colloidal gold as the labeling marker to visualize the location of antibodies and nucleotide probes that have been bonded to specific antigens and nucleotide sequences in tissue sections or cytologic preparations. The initially tiny gold particles are highly uniform in size and are enlarged further by AMG, thereby precipitating metallic silver onto the gold label surface. Under the light microscope (LM), the enlarged particles are black and provide a highly contrasting colored marker, standing out distinctly against the unstained background structures. Under the electron microscope (EM), the gold or gold-silver particles are highly electron-dense and provide a clear marker for target detection at the ultrastructural level. Gold-silver staining is one of the few procedures that can demonstrate the same target molecules at both the LM and EM levels. It is one of the most sensitive techniques available today for low copy number antigen and nucleotide detection. This unique technique has tremendously enhanced the scientific field of molecular morphology.
The detecting sensitivity of any immunostaining or in situ hybridization technique depends to a large degree on the signal-to-noise ratio. The larger and more visible the labeling signals are against the same background staining, the more sensitive the technique is. The ultimate achievement of these methods is to detect a single copy of a nucleotide sequence or a single molecule of an antigenic epitope on routinely processed tissue sections. In combination with tyramide signal amplification and comparable setups of label multiplication, gold-silver staining has more or less achieved this goal.
In spite of the tremendous power and potential of AMG and gold-silver staining, its merits and applications have not been widely recognized. Different morphological labeling methods are being used by many laboratories; however, gold-silver staining is exceptional in many ways. The small unified sizes of the gold particle can be controlled and can facilitate different degrees of tissue penetration. The degree of particle enlargement by silver or gold salt is also controllable so that to some degree the detecting sensitivity can be adjusted. Gold-silver staining is easy to perform, and the reagents required are readily available. The black color of the end product provides distinct advantages for fast screening of the preparations; the particularity of the stain provides a very sharp and brilliant image. These and other merits of this technique make it a favorite choice of many scientists.
The frontier of pathology is effectively moving into the age of molecular morphology. Simple morphologic patterns, provided by routine histochemical stains alone, are no longer adequate for depicting the subtle distinction between normal and diseased states, that can make significant differences in prognosis and therapy. Against this background, our goal is to provide the readers with the latest information about this extremely sensitive approach to microscopically localizing minute quantities of proteins or peptides and genetic material.
This volume gives a timely overview and detailed description of different approaches and applications in chapters written by the leading authorities in this emerging field. The authors include such scientists as those who made the landmark contributions to the development of this technique. LM and EM silver staining, AMG, and heavy metal detection are described by two living legends and their collaborators, Lars Grimelius and Gorm Danscher. The inventors of clustered gold covalently bound to macromolecules, Jim Hainfeld and collaborators, describe alternative techniques of silver and gold enhancement as well as their outstanding Nanogold® reagent and its many uses. Immunohistochemical and molecular morphological immunogold-silver staining are reviewed by Gerhard Hacker and Raymond R. Tubbs, both of whom are long-time advocates of the technique. Its original inventor, Osamu Fujimori, discusses his protein A-gold-silver staining method. Rick Powell presents a very promising reagent that combines a fluorescent label with gold-silver technology. Peter Jackson, one of the original inventors of immunogold-silver staining, introduces a new in situ hybridization method using a thermocycler designed for in situ polymerase chain reaction (PCR). Adalberto Merighi and his outstanding group of transmission electron microscopists contribute a chapter on ultrastructural applications of immunogold-silver staining. Hajime Sawada, John M. Robinson, and coauthors describe instructive protocols on pre- and post-embedding immuno-EM using Nanogold. Christian Schöfer and Klara Weipoltshammer, pioneers of EM tyramide signal-amplified gold-silver staining, instruct the reader about supersensitive EM immunocytochemistry and in situ hybridization. Finally, Bao-Le Wang and Jozef ˇSamaj and coworkers discuss applications of gold-silver scanning electron microscopy in cancer research and other fields.
The chapters in this book offer a balanced view of this emerging field. At the same time, they enable the readers to follow step-by-step protocols to make these procedures routine methods in their laboratories. This method allows the visualization of molecules that have never been localized before. With it, new discoveries follow naturally.
The editors feel that far too many animals are being used for experimentation, and very often only to fulfill legal requirements, to retest already evaluated drugs, or simply to reach academic titles and degrees. It is the explicit wish of the editors to set an example with this book. Responsible researchers should learn to avoid such experiments whenever they can. Most contributors responded to our urge and used human tissue or cell cultures in conducting experiments whenever possible. We wish to thank them for their kindness towards our friends in the animal kingdom.