IMPROVED MESSENGER-RNA IN-SITU HYBRIDIZATION ON FORMALDEHYDE-FIXED AND PARAFFIN-EMBEDDED TISSUE USING SIGNAL AMPLIFICATION WITH DIFFERENT HAPTENIZED TYRAMIDES

We report an optimized in situ hybridization (ISH) protocol with a rap id signal amplification procedure based on catalyzed reporter depositi on (CARD) to increase the sensitivity of non-isotopic mRNA ISH on form aldehyde-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 av ailable and newly synthesized haptenized tyramides, including digoxige nin-, biotin-, di- and trinitrophenyl- as well as fluorescein-tyramide , were compared. The haptenized tyramides were visualized using peroxi dase conjugated anti-hapten antibodies followed by the diaminobenzidin e reaction. As a test system, we applied digoxigenin-labeled oligonucl eotides to detect insulin and vasoactive intestinal polypeptide mRNA i n pancreatic endocrine tumors and liver metastases. Our results indica te that specificity, sensitivity, and applicability of oligonucleotide mRNA ISH can be significantly improved by using chemically digoxigeni n-labeled oligonucleotide probes and signal amplification by CARD. Fur thermore, all tested tyramides provided approximately equal amplificat ion efficiency. In conclusion, CARD signal amplification should furthe r promote mRNA ISH studies on paraffin-embedded tissues and allow for multiple-target nucleic acid detection in situ.