HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL DETECTION OF NEURONS THAT PRODUCE NITRIC-OXIDE - EFFECT OF DIFFERENT FIXATIVE PARAMETERS AND IMMUNOREACTIVITY AGAINST NONNEURONAL NOS ANTISERA

This study focused on two points concerning the histochemical and immu nohistochemical detection of neurons that produce nitric oxide (NO): ( a) the effect of fixation and other methodological parameters on the s taining pattern of both NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, and (b) the possibil ity that neurons display immunoreactivity against NOS antisera obtaine d from non-neuronal sources. Frontal sections of rat brains, fixed wit h 4% paraformaldehyde according to different protocols, were processed for single and double labeling using NADPH-d histochemistry and neuro nal (nNOS), macrophagic (macNOS), and endothelial (eNOS) NOS immunohis tochemistry. Our results show that variations in the fixative schedule , even within standard parameters, produce qualitative and quantitativ e changes in NADPH-d labeling. The effect of fixative on weakly staine d neurons is different from that on heavily stained neurons. In subfix ed brains, a large number of NOS-positive neurons lose their NADPH-d a ctivity whereas NOS immunolabeling remains unaltered. This finding may be particularly interesting in morphological studies that compare NAD PH-d activity under experimental conditions that can affect brain perf usion. On the other hand, many cortical and subcortical neurons show m acNOS immunoreactivity, most of it colocalized with nNOS.