Fixation conditions affect the intensity but not the pattern of NADPH-diaphorase staining as a marker for neuronal nitric oxide synthase in rat olfactory bulb.

Abstract

NADPH-diaphorase (NADPH-d) is commonly used as a histochemical marker for the neuronal form of the enzyme nitric oxide synthase (NOS). A recent biochemical study showed that in broken-cell preparations NADPH-d activity did not fully represent NOS and that NOS-unrelated NADPH-d activity was suppressed during fixation. Because it is unknown whether fixation also affects NOS-associated NADPH-d activity, we investigated the effects of various widely used fixatives on NADPH-d staining in relation to NOS immunoreactivity, obtained with polyclonal antibodies, in rat olfactory bulb. We found that the intensity of NADPH-d staining associated with NOS, as well as that unrelated to NOS, depends on fixation conditions. Addition of glutaraldehyde or lysine/sodium periodate to the fixative decreased intensity of NADPH-d staining. Fixative-dependence of NADPH-d staining was observed not only in the presence of the "normal" co-substrate beta-NADPH but also in the presence of the stereoisomer alpha-NADPH. Unlike the staining intensity, the staining pattern of NOS-associated as well as NOS-unrelated NADPH-d did not change after treatment with various fixatives. Our findings are of considerable practical significance because it has become clear that fixation conditions affect the sensitivity but not the selectively of the NADPH-d reaction as a marker for the presence of NOS.