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 immunohistochemistry represent a compromise between sensitive detection of the tracer and the immunohistochemical procedure. Therefore, it was investigated whether the use of tyramide amplification allows sensitive anterograde tracing with wheat-germ agglutinin conjugated to horseradish peroxidase (WGA–HRP) in conjunction with simultaneous immunohistochemistry. Vagal afferents were anterogradely labeled by injection of WGA–HRP into the nodose ganglion of rats. By use of tyramide–biotin amplification, a dense fiber plexus of vagal afferents was visualized centrally in the nucleus of the solitary tract and in retrogradely labeled neurons in the dorsal vagal nucleus. In the esophagus and duodenum, large- and small-caliber vagal fibers and terminals could be demonstrated comparably to conventional tracing techniques using carbocyanine dyes or WGA–HRP and TMB histochemistry. Combination with immunohistochemistry could easily be done, requiring only one more incubation step, and did not result in loss of sensitivity of the tracing. With this method and con-focal microscopy, the presence of Ca binding proteins in vagal afferent terminals could be demonstrated. Tyramide amplification allows sensitive anterograde tracing with low background staining in conjunction with immunohistochemistry of intra-axonal markers.

A Nonradiometric Detection System For the Rapid and Easy Detection of Oxytocin Oligonucleotide Probes in In Situ Hybridization

We describe the easy and rapid detection of oxytocin oligonucleotide probes in in situ hybridization using a biotinyl tyramide amplification system (TSA).There are several some instances where related proteins have sequence homology so great that only oligonucleotide probes will allow one to distinguish between their mRNAs. Vasopressin and oxytocin are such a pair. Traditional in situ hybridizationusing 35S labeled probes allows the specific detection of oxytocin mRNA.See FIG. 1.Nonradiometnc detection of oligonucleotide probes can be very difficult since the amount of labeling possible is limited by the small size of the probe. Traditional nonradiometric detection of a 30-mer biotin-labeled oxytocin probe with streptavidin-HRP followed by DAB detection proved essentially negligible. See FIG. 2.A novel tyramide amplification system (TSA) was used to increase the detection of the oxytocin probe signal. Most of the protocol was unchanged from the standard nonradioactive methodology. The biotin-labeled probe was hybridized overnight as usual. Stringency washes were also done as usual the next morning.