Immunocytochemical detection of neuronal NO synthase in rat brain cells

The aims of the present work were to identify the neuronal form of nitric oxide synthase (nNOS type I) in brain structures in rats by immunocytochemistry, to compare the results with data from histochemical reactions for NADPH-diaphorase, and to develop the optimal conditions for fixation for detecting nNOS. The product of the histochemical reaction was found to be located strictly in the cytoplasm. Immunocytochemical detection of nNOS showed that along with the cytoplasmic reaction for nNOS, the nuclei of some neurons and gliocytes were immunopositive, though the cytoplasm of these cells gave negative reactions for nNOS. Selection of the optimal fixation conditions for specimens and the dilution of the primary antibody allowed reductions in the intensity of nuclear nNOS-type reactions without affecting the specific reaction of the cytoplasm for nNOS. These data provide evidence that the best detection of nNOS in paraffin sections is obtained using immersion fixation in Carnoy's fluid or post-fixation in this solution after perfusion with 4% paraformaldehyde.