When NADPH diaphorase (NADPHd) works in the presence of formaldehyde, the enzyme appears to visualize selectively cells with constitutive nitric oxide synthase (NOS)

The NADPH diaphorase (NADPHd) reaction for nitric oxide synthase (NOS) visualization suffers from the circumstance that the diaphorase activity of NOS represents only part of the total diaphorase activity, and so far all efforts to make the reaction more specific for routine studies failed. The present investigation describes a simple procedure for mouse tissue, which allows the selective staining primarily of neurons, vascular endothelial cells and macula densa cells, those cells where constitutive NOS has been described reliably. In this method unfixed cryosections and 0.5 — 1% phosphate-buffered formaldehyde containing 0.5 mg NADPH/1 ml and 1 mg nitro BT/1 ml are used. Compared to strong prefixation with formaldehyde after which many additional cells are still positive for NADPHd, presence of formaldehyde in the incubation medium obviously allows the selective reaction of NOS-positive cells. In conclusion, compared with the original technique a more specific method for the visualization of the NADPHd activity of NOS appears to be available now and can be used for NOS studies in all kinds of mammalian species.