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Microscopy and stains in histology
Immune sera, Antibodies
Markers and conjugates
Tissue preparation
Immuno-staining, other ligand detection
Selection of staining protocols
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HomeCell-MarkerImmuno-staining, other ligand detection → Staining enhancement and signal amplification

Staining enhancement and signal amplification

Wolf D. Kuhlmann

Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany

Several methods exist to enhance immunological signals in immunohistology. One of such enhancement methods is tyramine amplification. The use of peroxidase to catalyze the deposition of phenols as principle for signal amplification in immunoassays was originally reported by MN BOBROW et al. (1989). This signal amplification method was called catalyzed reporter deposition (CARD). The tyramine signal amplification by catalized reporter deposition (CARD) was adapted to immunohistology and in situ hybridization in order to increase the sensitivity of antigen and DNA detection, respectively (ADAMS JC 1992). Another enhancement strategy is based on a reagent in which multiple molecules of linking antibodies (secondary antibodies) and marker enzyme are conjugated to a polymer backbone. This virtually background-free polymer-based detection sytem allows up to fivefold higher dilutions of primary antibodies than the ABC, LSAB or APAAP methods (SABATTINI E et al. 1998; VYBERG M and NIELSEN S 1998). Then, a quite new approach for the amplification of immunological signals is to combine the CARD amplification potential with ultrasmall gold probes and the silver intensification method or to use enzyme metallography (HAINFELD JF and POWELL RD 2000; MAYER G et al. 2000). In this way, a targeted enzyme conjugate mediates the deposition of metal from a solution to give dense, sharply defined signals. For example, HRP conjugates are used as probes. The enzymatic activity of HRP catalyzes the selective deposition of metal from a solution (an appropriate metal source, e.g. silver ions, and activating agents) at the site of the targeted substance. Furthermore, other enhancing methods are described such as rolling circle amplification (immuno-RCA; SCHWEITZER B et al. 2000; GUSEV Y et al. 2001) and post-chromogenic enhancements with cobalt and nickel salts.