2.6. DHE Staining for eNOS Uncoupling

For identifying eNOS as a source of superoxide, aortic rings were incubated in L-NAME (500 μM) for 30 mins at 37°C before freezing for cryosectioning as previously described [21]. Cryosections were incubated with the superoxide-sensitive fluorescent dye dihydroethidium (DHE (1 μM), Molecular Probes) in a humidity chamber for 30 mins at 37°C. Cover slips were then placed on the slides and kept in the dark for 20 mins. Fluorescence was detected (absorbance: 518 nm, emission: 605 nm) using Olympus BX61 microscope with a RetigaEXi camera (QImaging, Surrey, Canada) and images were analyzed using corrected total cell fluorescence (CTCF) method using ImageJ software (NIH, Bethesda, MD).