Tyramide biotinylated

Lebaron, P. Catala, P. Fajon, C. Joux, F. Baudart, J. Bernard, L. A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and tyramide signal amplification. Appl. Environ. Microbiol. 1997, 63, 3274-3278. 

FIGURE 11.13 Formation of reactive biotinylated tyramide through the catalytic reaction HRP and hydrogen peroxide catalyses. Source www.hmds.org.uk/histology.html... 

Signal amplification incubate sections for 8 10 min at room temperature with biotinylated Tyramide in amplification diluent (0.02% H202 in PBS) dissolved 1 1,500 to achieve the final concentration of the active tyramide 3.87 pM and wash in PBS for 2x3 min. 

This method, either standard or elite (increased molar ratio), remains the forefront of much of the immunocytochemistry being performed today, and will be the main subject of this chapter. There are new methods, though, that are being used with increased frequency, such as the labeled-avidin binding method, sometimes called the streptavidin-binding method, and a newer catalyzed amplification method that uses avidin, biotin, peroxidase, and a biotinyl tyramide to achieve even more sensitivity. These methods will be discussed at the end of this chapter. 

For the procedure, add the biotinyl tyramide (Dupont NEN, Boston, MA, or Dako, Carpinteria, CA) solution in place of the DAB solution in Subheading 3.1., step 19 and incubate for 15 min. Rinse as in Subheading 3.1., step 16. 

As the TSA-ABC protocol dramatically improves the signal intensity by the peroxidase-catalyzed deposition of biotinylated tyramide, blocking of endogenous peroxidase is required. To ensure quenching of the residual peroxidase, the use of a higher concentration and duration of treatment with H202 is recommended. 

Gold labeling may be used in the recently introduced catalyzed reporter deposition-immunogold technique where biotinylated tyramide molecules are attached the antibody-antigen complex site the biotinylated sites are visualized by interaction with streptavidin-gold (46,47). 

The tyramide amplification technique is based on the ability of phenolic compounds to become oxidized to highly reactive and unstable intermediates (8). When biotinyl tyramide is oxidized, dimerization with electron-rich aromatic compounds, such as those found in protein molecules, occurs (9). This reaction can be harnessed in immunohistochemistry to generate highly reactive biotinyl-tyramide intermediates that bind rapidly to protein molecules in the immediate vicinity of peroxidase enzymes. This reaction results in the deposition... 

The sequence of streptavidin-peroxidase and biotinyl-tyraminde can be alternately applied to perform a cycled tyramide amplification procedure. In practicality, however, cycling usually cannot exceed two or three cycles before background staining limits the utility of this approach. Commercial tyramide amplification products are available and include Tyramide Signal Amplification (TSA, DuPont NEN Life Sciences, Boston, MA) and Catalyzed Signal Amplification (CSA). ... 

Optional step is to amplify the signal with a biotinylated phenolic compound (e.g., biotinyl tyramide) for 15 min followed by a PBST wash. Biotinyl tyramide is included as part of the CSA Dako kit, although our experience with the Bcl-2 family of proteins has shown that this step is not necessary. 

Key words Avidin, Biotin, Avidin-biotin complex (ABC), Labeled avidin binding (LAB), Biotinylated tyramide, TBS buffer, DAB chromogen... 

Fig. 3. Diagram illustrating the amplified biotin substrate method. The biotinyl tyramide compound TSAforms biotin when oxidized by peroxidase. The deposition of biotin in the vicinity of the original ABC reagent allows for further amplification by the addition of peroxidase-labeled avidin or more ABC, followed by substrate (immunoglobulin biotin A avidin peroxidase).

Yang S, Witkowski A, Hutchins RS, Scott DL, Bachas LG (1998) Biotin-modified surfaces by electrochemical polymerization of biotinyl-tyramide. Electroanalysis 10 58-60... 

FreedmanLJ, MaddoxMT (2001)Acompari-son of anti-biotin and biotinylated anti-avidin double-bridge and biotinylated tyramide immunohistochemical amplification. J Neurosci Methods 112 43 9... 

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