Immunocytochemistry of endothelial nitric oxide synthase in the rat brain: a light and electron microscopical study using the tyramide signal amplification technique

There are many inconsistencies in the literature about the cellular and subcellular distribution of the endothelial isoform of nitric oxide synthase (eNOS) in the brain. We have re-investigated its localization by light and electron microscopical (LM, EM) immunocytochemistry and the NADPH-diaphorase reaction. Using bovine aortic tissue as a positive control the protocols for the fixation and staining procedure were optimized. Only cryosections immersion-fixed with aceton and a mixture of aldehydes exhibited a clear-cut immunostaining. In rat brain tissue the endothelium of the entire vasculature showed immunoreactivity and, in addition to that, the epithelial cells of the choroid plexuses, whereas neurons never displayed any signs of immunostaining. EM immunoprecipitates were seen irregularly distributed in the cytosol or attached to endocellular membranes. EM NADPH-diaphorase histochemistry using the tetrazolium salt BSPT provided incoherent pictures in so far as the reaction product was exclusively bound to membranes. The restriction of eNOS within brain tissue to the vasculature may have implications for the differential significance of NOS isoforms in brain function.