Nitric oxide detection by DAF (diaminofluorescein) fluorescence in human myometrial tissue

Nitric oxide (NO) is considered to be involved in the modulation of uterine contractility. In the present pilot study, the direct detection of intracellular NO in pregnant human myometrial tissues has been investigated by using the fluorescent indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA). Pregnant myometrial tissue samples were obtained during Cesarean sections between week 34 and 40 of gestation before the onset of labor. Living explants were loaded with 10 microM DAF-2DA, stimulated with 1 mM arginine, subsequently fixed with glutaraldehyde and examined by fluorescence microscopy. The presence of NO synthases (NOS) was studied by immunohistochemistry. After application of DAF-2DA, DAF fluorescence was located primarily in blood vessels and to a minor extent in myometrial cells. By immunohistochemistry, strong endothelial NOS (eNOS) staining was found in vessel walls. In myometrial cells weak staining of eNOS and inducible NOS was observed. We conclude that the direct NO detection by DAF-2DA provides a new and independent method to identify sites of NO production in myometrium and other heterogeneous tissues.