Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining

The discovery of immediate early genes (IEG) has provided neuroscientists with a new functional mapping technique. Labelling of neural tissue for the protein product of IEG provides an activity map with single-cell resolution. When combined with labelling for the chemical identity of the neuron, this provides a powerful tool for the investigation of specific cell populations along a neuraxis. Here we describe in detail a method which allows simultaneous bright-field visualization of neurochemically identified cells displaying increased IEG expression. This technique is evaluated in tissue from rats subjected to stimuli known to induce the expression of the IEG c-fos in various medullary catecholaminergic and hypothalamic neurosecretory cell groups. A 2-colour immunoperoxidase technique was used to visualize Fos, the nuclear protein product of c-fos, and the cytoplasmic antigens tyrosine hydroxylase (TH), phenylethanolamine N-methyl transferase (PNMT), oxytocin (OT) and vasopressin (VP). This involved simultaneous application of primary antibodies raised in different species followed by sequential application of appropriate biotinylated secondary antibodies and the avidin-biotin-peroxidase technique. Fos was visualized with nickel-intensified diaminobenzidine (Ni-DAB) in the first sequence while TH, PNMT, OT or VP were visualized with DAB alone, resulting in readily distinguishable black and amber reaction products, respectively. This dual immunoperoxidase technique is time saving compared to techniques using sequential application of primary antibodies and avoids the disadvantages associated with fluorescence techniques.