Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method

A simple technique is described for simultaneously visualizing the cells of origin of descending brain stem-spinal systems and immunocytochemically defined neurotransmitters. The combination of horseradish peroxidase (HRP) histochemistry, enhanced by the use of CoCl 2, with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunocytochemical staining method, revealed double labeled cells containing black punctate HRP granules within a homogeneous brown cytoplasm stained positively for serotonin (5-HT), substance P or enkephalin-like immunoreactivity. The advantages of this method over other double labeling techniques are discussed.