En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

The overall goal of this procedure is to simultaneously detect intracellular nitric oxide and superoxide anions using the fluorescence dye DAF-2DA and DHE in freshly isolated intact mouse aortas. This method can help in lab recovering researcher's case in the cardiovascular physiology field such as endothelial dysfunction and eNOS uncoupling. The main advantage of this technique is to provide a simple and precise method for ex vivo direct detection and visualization of nitric oxide and superoxide anion simultaneously in intact blood vessels.

For this protocol, construct an organ bath system which can be heated to 37 degrees Celsius and aerated with 95%oxygen, 5%carbon dioxide gas from a carbon gas tank. To begin, prepare enough fresh Krebs-Ringer Bicarbonate Buffer as needed to bathe the tissues of interest. For example, 200 milliliters is needed for aortas in one experiment.