Tyramide signal amplification

HRP Horseradish Peroxidase 1st AB - Primary Antibody 2nd AB - Secondary Antibody 

Active ester stock solution A dissolvelO mg of 5-(and-6)carboxyfluorescein, succinimidyl ester (FITC-NHS, Molecular Probes -1311) in 1 ml of DMF. 

Tyramine-HCl stock solution B dissolving 10 mg of Tyramine-HCl (Sigma, T-2879) in 1 ml of DMF to which 10 pi of triethylamine (TEA, Pierce) was added. 

For efficient acylation, mix 340 pi of stock solution B with 1 ml of stock solution A, and leave at room temperature in the dark for 2 h. 

The synthesized tyramide conjugates are diluted in 10 ml 100% methanol. The tyramide stock solutions may be safely stored for at least 8 months at 4°C without any reduction in reactivity. 

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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. 

Ness, J.M., Akhtar, R.S., Latham, C.B., and Roth, K.A. (2003) Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection. J. Histochem. Cytochem. 51, 981-987. 

Signals obtained by methods like tyramide signal amplification can enhance signal intensity, reduce background fluorescence, and then increase sensitivity. 

Fig. 6.2 Tyramide signal amplification. T is the labeled tyramine and HRP, horse radish peroxidase. The Label can be a lluorochrome or biotin. The lluorochrome can be visualized directly in a fluorescence microscope. Biotin can be visualized via labeled streptavidin...

Tyramide signal amplification This procedure, designated as a catalyzed reporter deposition (CARD) or tyramide signal amplification (TSA), takes advantage of horseradish peroxidase (HRP) from an HRP-labeled secondary antibody to catalyze in the presence of hydrogen peroxide the oxidation of the phenol moiety of labeled tyramine. On oxidation by HRP, activated tyramine molecules rapidly bind covalently to electron-rich amino acids of proteins immediately surrounding the site of the immunoreaction. This allows an increase in the detection of an antigenic site up to 100-fold compared with the conventional indirect method with no loss in resolution. 

Tyramide signal amplification (TSA PerkinElmer Life Sciences, Boston) and enzyme-labeled fluorescence (ELF Molecular Probes) are related detection technologies. In the tyramide amplification process, a tyramide-biotin complex is produced by the action of horseradish peroxidase. The complex precipitates near the binding site and accumulates. The complex is detected by the use of streptavidin-Cy3/Cy5. 

Karsten SL, Van Deerlin VM, Sabatti C et al. An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis. Nucleic Acids Res 2002 30 E4. 

Wiedorn KH, Kuhl H, Galle J et al. Comparison of in situ hybridization, direct and indirect in situ PCR as well as tyramide signal amplification for the detection of HPV Histochem Cell Biol 1999 11 89-95. 

Qian X, Bauer RA, Xu HS et al. In situ hybridization detection of calcitonin mRNA in routinely fixed, paraffin-embedded tissue sections a comparison of different types of probes combined with tyramide signal amplification. Appl Immunohistochem MolMorphol 2001 9 61-69. 

Extensive progress has been attained in determination techniques. Noteworthy is the broad applicability of a homogenic method known as the TRACE method (time-resolved amplified cryptate emission). Another interesting solution, TSA (tyramide signal amplification), employs a technique which amplifies the tyramide signal (Bednarski and Reps, 2003). 

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). ... 

In tyramide signal amplification (TSA), the target cells are labelled with an antibody or a nucleic acid probe, followed by secondary detection with a horseradish peroxidase (HRP) labelled antibody. HRP activates multiple copies of fluores-cently labelled tyramide derivatives, yielding fluorescent tyramide radicals that are deposited in the vicinity of the HRP-target interaction site (Fig. 5). 

Toda Y, Kono K, Abiru H, et al. Application of tyramide signal amplification system to immunohistochemistty A potent method to localize antigens that ate not detectable by ordinary 122. method. Pathol Int. 1999 49 479. 

Finally, there are numerous techniques for signal enhancement such as avidin - biotin - complexes and tyramide signal amplification methods. All of these tertiary procedures obviously add to the time parameter of any test, but can add significant staining intensity that is critical in samples with low numbers of antibody binding sites. Determination of methodology for cell staining must be evaluated on the basis of tissue/cells samples to be examined. 

Tyramide signal amplification (TSA) is a powerful amplification method that allows fluorescent labeling to be significantly amplified. TSA is a patented technology from Perkin Elmer that has been licensed to other companies for use in reagents. 

Fig. 7.9 Tyramide signal amplification (TSA) immunocytochemistry. The 1° antibody binds to the antigen and a 2° antibody labeled with HRP binds to the 1° antibody. The labeled tyramide reagent is added with a small amount of the catalyst H2O2. The enzyme HRP converts the inactive labeled tyramide into a reactive labeled tyramide that binds to the amino acid tyrosine found in proteins...

Tyramide signal amplification (TSA) - a powerful amplification method that uses labeled tyramide, which is activated by HRP and binds tyrosine amino acids on adjacent proteins. 

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