Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides |
| |
Authors: | E J M Speel Parvin Saremaslani Jürgen Roth Anton H N Hopman Paul Komminoth |
| |
Institution: | (1) 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, CH;(2) Department of Molecular Cell Biology & Genetics, University Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands, NL |
| |
Abstract: | We report an optimized in situ hybridization (ISH) protocol with a rapid signal amplification procedure 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 haptenized tyramides were visualized using peroxidase
conjugated anti-hapten antibodies followed by the diaminobenzidine reaction. As a test system, we applied digoxigenin-labeled
oligonucleotides to detect insulin and vasoactive intestinal polypeptide mRNA in pancreatic endocrine tumors and liver metastases.
Our results indicate that specificity, sensitivity, and applicability of oligonucleotide 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 amplification efficiency. In conclusion, CARD signal amplification should further promote mRNA
ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ.
Accepted: 1 July 1998 |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|