Signal Amplification in the Detection of Single-copy DNA and RNA by Enzyme-catalyzed Deposition (CARD) of the Novel Fluorescent Reporter Substrate Cy3.29-Tyramide

Abstract

SUMMARYWe demonstrate that the CA talyzed R eporter D eposition method (CARD), utilizing the novel fluorescent reporter Cy3.29-tyramide, is successful in the F luorescent I n

Amplification Methods to Increase the Sensitivity of In Situ Hybridization: Play CARD(S)

“…Also the use of viscosity-increasing polymers such as dextran sulfate or polyvinyl alcohol has been suggested for improved localization of tyramides (Van Gijlswijk et al 1996a), which, however, proved not to be essential in our hands (see Figure 2). An even more rapid way to visualize nucleic acids in situ is the use of peroxidaselabeled probes, which can be detected directly with CARD signal amplification (Van Gijlswijk et al 1996b;Schmidt et al 1997). However, these procedures proved to be clearly less sensitive than the indirect procedures described above, although here the application of a second signal amplification reaction after detection of the first layer of deposited haptenized tyramides by a peroxidase-labeled anti-hapten antibody conjugate may be advantageous to further increase the detection sensitivity.…”

Sensitive Multicolor Fluorescence In Situ Hybridization Using Catalyzed Reporter Deposition (CARD) Amplification

“…It has been established that the highly reactive intermediates of this reaction will bind to tyrosine moieties of proteins present in the immediate vicinity of the peroxidase binding site. Because horseradish peroxidase conjugates are commercially available for use in many biochemical assays, the CARD system has been easily implemented not only in immunocytochemistry (Adams et al 1992;Berghorn et al 1994;Merz et al 1995) but also in DNA and RNA detection procedures using FISH (Kerstens et al 1995;Raap et al 1995;Macechko et al 1997;Schmidt et al 1997). Despite the high sensitivity that could be obtained in these single-target FISH experiments, the discreteness of FISH signals was often limited.…”

Biotinyl–Tyramide: A Novel Approach for Electron Microscopic Immunocytochemistry

“…This deposition is believed to take place at the site of the enzyme reaction, thus leading to good resolution (Bobrow et al 1992). The development of fluorescent tyramide reagents has made possible the application of this technique to fluorescent microscopy, either in immunostainings (Berghorn et al 1994;De Haas et al 1996;Hunyady et al 1996;Plenat et al 1997;van Gijlswijk et al 1997;Van heusden et al 1997) or in in situ hybridization (Kerstens et al 1995;Raap et al 1995;De Haas et al 1996;Macechko et al 1997;Schmidt et al 1997;van Gijlswijk et al ,1997Zehbe et al 1997).…”