A nonradioactive method for localization of endothelin receptor mRNA in situ

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 ET A and ET B 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, ET A receptor mRNA was localized to the medial smooth muscle layer and ET B 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 ET B receptor mRNA in the scarred and infarcted zone of the left ventricle. This method provides a suitable altemative 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.