The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3'-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level.
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