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Tyramide Signal Amplification (TSA) is easily integrated into any protocol after an initial addition of horseradish peroxidase (HRP). The HRP is used to catalyze the deposition and binding of a labeled (e.g. biotin or other labeling moieties) tyramide onto tissue sections or cell preparations previously blocked with proteins. This reaction is quick (less than 10 minutes) and the binding is covalent because the reaction intermediate dimerizes with tyrosine residues on the surface-bound endogenous proteins. These labels can then be detected by standard chromogenic or Fluorescent techniques. Since the added labels are only deposited proximal to the enzyme site, there is minimal, if any, loss of resolution.
Detection Alternatives using TSA
TSA for direct fluoresence detection
- Choice of five different fluorophores--Fluorescein, Tetramethylrhodamine, Coumarin, Cyanine 3, and Cyanine 5
- Direct detection of fluorescence signal immediately after the amplification step
- Chromogenic detection is possible using TSA fluorescein system. The fluorescein tyramide amplification step is followed by an anti-fluorescein enzyme conjugate and then visualized with the appropriate chromogen.
- Ideal for multi-target detection
| Comparison of Direct Immunofluorescent Staining and TSA enhanced Direct Fluorescent staining of CMV Infected Cells. |
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| Direct Fluorescent Staining |
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Direct Fluorescent staining enhanced by TSA-Direct |
TSA and TSA Plus for chromogenic detection
- Choice of two haptens-biotin or DNP
- Deposited biotin or DNP labels visualized with standard enzymes (HRP or alkaline phosphatase) and chromogens (DAB, AEC, BCIP/NBT, etc.)
- A streptavidin-fluorophore conjugate, such as SA- Fluorescein, SA-Texas Red®, and SA-Coumarin can be used for fluorescence signal generation.
- Provides maximum signal amplification for low abundance targets
| IHC detection of CD3 antigen in serial sections of paraffin-embedded human tonsil. Courtesy of F. van den Berg and A. de Koning, Dept. Pathology, AMC, Amsterdam, The Netherlands. |
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| Rabbit anti-CD3 1:400, biotinylated anti-rabbit, ABC, with DAB chromogenic detection. |
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TSA Biotin: Rabbit anti-CD3 1:400, biotinylated anti-rabbit, SA-HRP, biotinyl tyramide, ABC, with DAB chromogenic detection. |
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