Light microscopic triple-colored immunohistochemical staining on the same vibratome section using the avidin-biotin-peroxidase complex technique.

The sequential application of the avidin-biotin-peroxidase complex technique was used to localize multiple tissue antigens on a single free floating section of rat brain. Sequential visualization of individual antigens was achieved by the silver-gold-intensified diaminobenzidine (DAB) in the first step, nickel-intensified DAB in the second step, and the DAB alone in the third step of the immunostain procedure. For the demonstration of this method, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF), and vasopressin (VAS) antisera were used. Sections from the hypothalamic paraventricular nucleus (PVN) of rats pretreated with colchicine were stained. Black TH containing cell bodies were clearly distinguished from blue stained CRF cells and from yellow stained VAS-containing cell bodies in the PVN on the 25-30 micron thick vibratome sections. The sequential immunostaining procedure presented here results in superior staining of multiple antigens as compared to that achieved by the sequential application of the peroxidase-antiperoxidase (PAP) technique.