&p.1:Abstract We report an optimized in situ hybridization (ISH) protocol with a rapid signal amplification proce- dure based on catalyzed reporter deposition (CARD) to increase the sensitivity of non-isotopic mRNA ISH on formaldehyde-fixed and paraffin-embedded tissue. The CARD method is based on the deposition of haptenized tyramide molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. Commercially available and newly synthesized haptenized tyramides, including digoxigenin-, biotin-, di- and trinitrophenyl- as well as fluorescein-tyramide, were compared. The hap- tenized tyramides were visualized using peroxidase con- jugated anti-hapten antibodies followed by the diamino- benzidine reaction. As a test system, we applied digoxi- genin-labeled oligonucleotides to detect insulin and va- soactive intestinal polypeptide mRNA in pancreatic en- docrine tumors and liver metastases. Our results indicate that specificity, sensitivity, and applicability of oligonu- cleotide mRNA ISH can be significantly improved by using chemically digoxigenin-labeled oligonucleotide probes and signal amplification by CARD. Furthermore, all tested tyramides provided approximately equal ampli- fication efficiency. In conclusion, CARD signal amplifi- cation should further promote mRNA ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ.&bdy: Introduction In situ hybridization (ISH) has proved to be an important tool in molecular cell biology and pathology for the de- tection and localization of specific mRNA sequences within cells and tissues (for reviews see Larsson and Hougaard 1990; Johnson et al. 1991; Pringle 1995; Dirks 1996; McNicol and Farquharson 1997). In particular, the introduction of non-radioactive probe labeling and detec- tion procedures has greatly advanced the feasibility and efficiency of mRNA ISH both in research and diagnostics (Larsson and Hougaard 1990; Komminoth et al. 1992; Hopman et al. 1995; Pringle 1995; Speel et al. 1995; Komminoth 1996). However, current mRNA ISH techniques often suffer from low sensitivity, poor resolution, and are time con- suming. Hence, improvements are required to permit rapid detection of mRNA sequences in routinely fixed, paraffin-embedded material. Several attempts have been made to increase the sensitivity of non-radioactive ISH procedures, such as mixtures of labeled, non-overlapping oligonucleotides (Lloyd and Jin 1995; Trembleau and Bloom 1995) or the application of up to five cytochemi- cal probe detection layers (Larsson and Hougaard 1990; Hamilton-Dutoit and Pallesen 1994; Smith et al. 1997). Recently, several signal and target amplification ap- proaches have been developed, including primed in situ DNA synthesis, in situ polymerase chain reaction, and the catalyzed reporter deposition (CARD) system (for a review see Komminoth and Werner 1997). The latter method, introduced by Bobrow et al. (1989), is based on the deposition of labeled tyramide molecules by means of peroxidase (HRP) activity. It has been established that the highly reactive intermediates formed during the HRP-tyramide reaction will bind to tyrosine-rich moi- eties of proteins present in the vicinity of the HRP bind- ing site. The CARD system has been implemented in im- munocytochemistry (Adams 1992; Berghorn et al. 1994; Merz et al. 1995) as well as in DNA and RNA detection procedures using fluorescence ISH (Kerstens et al. 1995; Raap et al. 1995; Schmidt et al. 1997; Speel et al. 1997a). E.J.M. Speel ( ✉ ) · P. Saremaslani · J. Roth · P. Komminoth Department of Pathology, Division of Cell and Molecular Pathology, University of Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland e-mail: ernst-jan.speel@pty.usz.ch Tel.: +41-1-2553030; Fax: +41-1-2554551 A.H.N. Hopman Department of Molecular Cell Biology & Genetics, University Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands&/fn-block: Histochem Cell Biol (1998) 110:571–577 © Springer-Verlag 1998 ORIGINAL PAPER &roles:Ernst J.M. Speel · Parvin Saremaslani · Jürgen Roth Anton H.N. Hopman · Paul Komminoth Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides &misc:Accepted: 1 July 1998