FIXATION CONDITIONS AFFECT THE INTENSITY BUT NOT THE PATTERN OF NADPH-DIAPHORASE STAINING AS A MARKER FOR NEURONAL NITRIC-OXIDE SYNTHASE INRAT OLFACTORY-BULB

NADPH-diaphorase (NADPH-d) is commonly used as a histochemical market for the neuronal form of the enzyme nitric oxide synthase (NOS). A rec ent biochemical study showed that in broken-tell preparations NADPH-d activity did not fully represent NOS and that NOS-unrelated NADPH-d ac tivity was suppressed during fixation. Because it is unknown whether f ixation also affects NOS-associated NADPH-d activity, we investigated the effects of various widely used fixatives on NADPH-d staining in re lation to NOS immunoreactivity, obtained with polyclonal antibodies, i n rat olfactory bulb. We found that the intensity of NADPH-d staining associated with NOS, as well as that unrelated to NOS, depends on fixa tion conditions. Addition of glutaraldehyde or lysine/sodium periodate to the fixative decreased intensity of NADPH-d staining. Fixative-dep endence of NADPH-d staining was observed not only in the presence of t he ''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 cha nge after treatment with various fixatives. Our findings are of consid erable practical significance because it has become cleat that fixatio n conditions affect the sensitivity but not the selectivity of the NAD PH-d reaction as a marker for the presence of NOS.