Posthybridization Signal Enhancement

6.2.1. Tyramide Signal Amplification A novel catalyzed reporter deposition (CARD) system originally described by Bobrow et al. in 1989 (57) has undergone considerable evolution in the last decade. This system is considered to enhance the ISH signal 2.5- to 100-fold with diverse modifications described in the literature. In principle, this system uses horseradish peroxidase (HRP) either conjugated to antibody against the hapten label of the ISH probe as seen in the original reports or linked directly to the nucleotides as found in the impressive experiments of Raap and his co-workers in recent years (58,59). The HRP catalyzes a covalent deposition of tyramide on tyrosine residues in the vicinity of enzyme action via a free-radical mechanism. The fluorescent labels of multiple tyramide residues thus deposited could be visualized by epifluorescence microscopy. An example of the tyramide amplification of ISH

signals for low-abundance cytokine mRNA species is shown in Fig. 4. In addition to its application for detecting low-copy-number mRNA or viral DNA, the tyramide amplification system could also be used for small LSI probes to specifically detect small chromosomal translocations. Despite the major limitation of this system resulting from the as yet sole dependence on peroxidase catalysis, the attempts to use multiple probes applied sequentially and employing HRP through different means, a multicolor FISH chromosomal analysis has been made possible in recent reports (59,60).

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