Biotinylated tyramine

Originally described several years ago for use in immunoassay systems (36), biotinylated tyramine amplification has recently been adapted for immuno-cytochemical use (37). The system is based on the ABC method. H2O2 is catalysed to H2O and oxygen-free radicals by HRP as usual. This then reacts with biotinylated tyramine to produce activated biotinylated tyramine. which binds to nearby proteins in a highly localized manner. The high concentrations of biotin thus produced can then be detected by the application of one additional layer of avidin-reporter conjugate. The system has been used to demonstrate antigens previously only detectable in frozen sections, and to increase primaiy antibody dilution. Several manufacturers produce kits that are based on the system (CSA from Dako Ltd. and TSA from NEN life Science Products), or home-made biotinylated tyramine can be produced. 

Add 100 mg of sulfosuccinimidyl 6-(biotinamido) hexanoate and 30 mg of tyramine-HCl to 40 ml of borate buffer, then stir gently overnight. 

Filter through paper, then through a 0.45 pm syringe filter, aliquot, and store frozen at -20 °C. 

Cany out standard ABC-based method Protocol 4) up to and including step 9.  

Wash the slides with TNT buffer three times for 5 min each. 

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Kim SH, Shin YK, Lee KM, Lee JS, Yun JH, et al. 2003. An improved protocol of biotinylated tyramine-based immunohistochemistry minimizing nonspecific background staining. J Histochem Cytochem 51 129-132. 

One hundred milligrams of sulfosuccinimidyl-6- (biotinimide) hexanoate (NHS-LC-biotin) (Pierce, Rockford, IL) is dissolved in 40 ml of 50 mM borate buffer (pH 8.0). To this solution is added 30 mg of tyramine hydrochloride (Sigma Chemical Company, St. Louis, MO). The solution is stirred overnight at room temperature and filtered (0.45 p,m filter). The final biotinylated tyramine concentration is 7 xM. Before application, the solution is diluted 1 160 in Tris-HCl buffer (pH 7.6) containing 0.03% H202. 

Kerstens, H. M. J., Poddighe, P. J., and Hanselaar, A. G. J. M. 1995. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem. 43 341-352. 

King G, Payne S, Walker F, et al. A highly sensitive detection method for immunohistochemistry using biotinylated tyramine. J. Pathol. 1998 183 237-241. 

Enhanced DAB by metal, imidazole, and so on CARD Enhance the color reaction HRP catalyzes deposition of biotinylated tyramine at the site of HRP ... 

Apply the biotinylated tyramine solution diluted 1 500 in Tris buffer containing 0.03% (v/v) H2O2. It may be necessary to process a number of slides with different incubation times between 30 sec and 10 min, as the resulting amplification can be quite variable. ... 

Enhancement of the DAB-HRP reaction product 405 The production and use of biotinylated tyramine 406... 

Originally described several years ago for use in immunoassay systems (36). biotinylated tyramine amplification has recently been adapted for immuno-cyiochemical use (37). The system is based on the ABC method. is catalysed... 

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