TYRAMIDE AMPLIFICATION ALLOWS ANTEROGRADE TRACING BY HORSERADISH PEROXIDASE-CONJUGATED LECTINS IN CONJUNCTION WITH SIMULTANEOUS IMMUNOHISTOCHEMISTRY

Current protocols for a combined approach of anterograde tracing with carbocyanine dyes or horseradish peroxidase (HRP) conjugates and immun ohistochemistry represent a compromise between sensitive detection of the tracer and the immunohistochemical procedure. Therefore, it was in vestigated whether the use of tyramide amplification allows sensitive anterograde tracing with wheat-germ agglutinin conjugated to horseradi sh peroxidase (WGA-HRP) in conjunction with simultaneous immunohistoch emistry. Vagal afferents were anterogradely labeled by injection of WG A-HRP into the nodose ganglion of rats. By use of tyramide-biotin ampl ification, a dense fiber plexus of vagal afferents was visualized cent rally in the nucleus of the solitary tract and in retrogradely labeled neurons in the dorsal vagal nucleus. In the esophagus and duodenum, l arge-and small-caliber vagal fibers and terminals could be demonstrate d comparably to conventional tracing techniques using carbocyanine dye s or WGA-HRP and TMB histochemistry. Combination with immunohistochemi stry could easily be done, requiring only one more incubation step, an d did not result in loss of sensitivity of the tracing. With this meth od and confocal microscopy, the presence of Ca binding proteins in vag al afferent terminals could be demonstrated. Tyramide amplification al lows sensitive anterograde tracing with low background staining in con junction with immunohistochemistry of intra-axonal markers.