Nearly three decades have passed since Faulk and Taylor first introduced colloidal gold as a label for immunoelectron microscopy (immuno EM) in 1971.23,55, 78 Since then, immunogold-staining (IGS) has gained widespread application by virtue of its various advantages over other, nonparticulate immunostaining techniques. As accumulations of colloidal gold in a concentration high enough to be seen as red color in the light microscope (LM) require the presence of very large amounts of antigen,28 introduction of silver enhancement (autometallography; AMG) by Danscher in 1981 became the prerequisite for obtaining a higher sensitivity in immunogold applications.13,14,16 Combination of this reaction with colloidal gold-

labeled enzyme histochemistry and immunohistochemistry (IHC) were introduced simultaneously in 1983 by Danscher et al.15 and Holgate et al.,53,54 respectively, and were considered real breakthroughs in immunogold technology. The new IHC technique evolving from this was named immunogold-silver staining (IGSS) and had a dramatically improved sensitivity and detection efficiency compared to the immunostaining techniques available at that time.37,88

As described in Chapters 2 and 3, the high sensitivity of IGSS is largely based on the high intensification potential of AMG (earlier misleadingly called “physical development”), applied to amplify the size of the gold particle(s) used as label of antibodies or streptavidin. Under the LM, conglomerations of gold particles embedded in silver appear as black precipitates with a

distinctly sharper appearance than the reaction products of most enzyme-labeled preparations. Silver lactate was originally used as the ion source in AMG,13,14 and size amplification of gold label is achieved by the formation of shells of metallic silver around each gold particle by the AMG process.18,44,46,61 The process is catalyzed by hydroquinone in a citrate buffer of low pH. Thereby, two surplus electrons present on each molecule of hydroquinone function as electron donors for the transformation of silver ions into metallic silver atoms. By precipitation of these metallic silver atoms onto (similarly sized) gold atoms on the surface of the gold particles, the latter ones are enlarged in size. If gold particles are sufficiently near to each other, which is the case when good immunolabeling is obtained, conglomeration of gold and silver takes place. Under the LM, gold-silver accumulations are clearly visu-

alized as grayish-black dots or areas and stand out well against the nonreacting background (Figures 4.1 through 4.5).