Key result
A nonradioactive in situ hybridization method using digoxigenin-labeled probes successfully localized ETA and ETB receptor mRNA in rat and human tissues, providing an alternative to radioisotopes.
A novel nonradioactive in situ hybridization method successfully localizes endothelin receptor mRNA in cardiovascular tissues, offering better tissue preservation and shorter detection times than traditional radioisotope methods.
Facilitates safer endothelin receptor mRNA localization in tissues; leaves open clinical utility in human cardiovascular disease.
To investigate relationships between the distribution of endothelin (ET) receptor expression and histopathology of heart and blood vessels, we developed a method of nonradioactive in situ hybridization in paraffin sections. Rat mesenteric bed, rat heart, and human uterine artery were fixed in formalin and embedded in paraffin ETA and ETB receptor cDNAs were subcloned into plasmid vectors for synthesis of sense and anti-sense probes. Digoxigenin (DIG)-UTP was incorporated into every twentieth to twenty-fifth nucleotide of the newly transcribed cRNA. mRNA was detected in situ using an anti-DIG alkaline phosphatase antibody and an alkaline phosphatase substrate. In blood vessels, ETA receptor mRNA was localized to the medial smooth muscle layer and ETB receptor mRNA to the endothelial and adventitial layers. Hearts from rats that had undergone coronary artery ligation for induction of CHF showed intense staining for ETB receptor mRNA in the scarred and infarcted zone of the left ventricle. This method provides a suitable alternative to radioisotope-labeled probes for detection of ET receptor mRNA. It allows better preservation of tissues, shorter detection time, and improved morphology for microscopic analysis.
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McEwan et al. (1998) studied this question. Nonradioactive in situ hybridization using digoxigenin-labeled cRNA probes vs. Radioisotope-labeled probes was evaluated on Localization of ETA and ETB receptor mRNA. A nonradioactive in situ hybridization method using digoxigenin-labeled probes successfully localized ETA and ETB receptor mRNA in rat and human tissues, providing an alternative to radioisotopes.
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