Dual immunostaining procedure demonstrating neurotransmitter and neuropeptide codistribution in the same brain section

A method for sequential immunostaining of two antigens in the same section and the application to neuronal elements in the CNS is described. This method employs the peroxidase-antiperoxidase (PAP) technique as a first sequence, followed by glucose oxidase-antiglucose oxidase (GAG) complex as the second sequence, yielding rich brown and vibrant blue reaction products, respectively. Application of the PAP/GAG immunoreagents as dual markers for various neuropeptide and neurotransmitter systems is described in detail and the procedure is outlined. Comparisons with other dual staining methods are discussed and the application to other systems is presented.