Aldehyde fixation differentially affects distribution of diaphorase activity but not of nitric oxide synthase immunoreactivity in rat brain

Abstract

The effect of aldehyde fixation on NADPH- and NADH-dependent diaphorase (d) histochemistry and nitric oxide synthase (NOS) immunocytochemistry in the brain was investigated by comparing the distribution of these enzymes in in situ nitrocellulose blots of unfixed brain sections with that in aldehyde-fixed brain sections. Substitution of NADPH by NADH yielded no gross differences in cellular distribution in the native blot, whereas in fixed sections NADH produced nonspecific staining of the entire section. In the in situ blot NADPHd histochemistry therefore visualized general nitroblue tetrazolium reductase (NBTr) activity, which was particularly strong in hippocampal pyramidal neurons and cerebellar Purkinje cells. Aldehyde fixation abolished the anatomical pattem of general NBTr activity and changed the histochemical distribution in that of the NADPHd activity associated with the distribution of NOS-I immunoreactivity (ir). Fixation intensified NADPHd histochemical staining in specific neurons, resulting in outstanding, Golgi-like staining of these neurons in several brain regions, whereas the general NBTr activity in pyramidal and Purkinje cells disappeared. In contrast to the histochemical diaphorase distribution, the distribution of NOS-I it on blots and in aldehyde-fixed brain sections was similar. No NOS was observed in hippocampal pyramidal and cerebellar Purkinje neurons. In regions like cerebral and cerebellar cortex and striatum the applied anti NOS-I serum had a higher affinity for the native protein. It is concluded that aldehydes, rather than to progressively suppress NOS-unrelated enzymes, differentially elicit NADPHd activity in some groups of neurons while leaving NOS-ir unaffected.