TSA™ Detection Kits

Invitrogen offers the Molecular Probes® TSA™ Detection Kits that combine the versatile tyramide signal amplification technology with our high-performance Alexa Fluor® dyes as well as our classic dyes like Oregon Green® and Pacific® Blue. This technology can also be used with colorimetric detection systems using our TSA™ biotin or DNP kits.  With TSA™ Detection kits from Invitrogen you get:

  • Ultrasensitive peroxidase-mediated signal amplification system
  • Immunohistochemical detection of low-abundance targets
  • Use of less antibody or hybridization probe
  • Minimized diffusion-related loss of signal localization

View all TSA™ Detection Kit products

What is Tyramide Signaling Amplification?

Tyramide Signal Amplification (TSA™) is an enzyme-mediated detection method that utilizes the catalytic  activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ.(1–5) TSA labeling is a combination of three elementary processes (Figure 1):

  1. Binding of a probe to the target via immunoaffinity (proteins) or hybridization (nucleic acids) followed by secondary detection of the probe with an HRP-labeled antibody or streptavidin conjugate.
  2. Activation of multiple copies of a fluorescent or biotinylated tyramide derivative by HRP.
  3. Covalent coupling of the resulting highly reactive, short-lived tyramide radicals to nucleophilic residues in the vicinity of the HRP–target interaction site, resulting in minimal diffusion-related loss of signal localization.

 

click for larger image

Figure 1—Schematic representation of TSA detection methods applied to immunolabeling of an antigen. The antigen is detected by a primary antibody, followed by a horseradish peroxidase–labeled secondary antibody in conjunction with a dye-labeled (or hapten-labeled) tyramide, resulting in localized deposition of the activated tyramide derivative (Stage 1). Further dye deposition, and therefore higher levels of signal amplification, can be generated by detecting dye deposited in stage 1 with a horseradish peroxidase–labeled anti-dye antibody in conjunction with a dye-labeled tyramide (Stage 2).

 

Tyramide Signaling Amplification in action:
  

Multicolor fluorescence analysis of mouse cerebellum cross section.

 

Fluorescence in situ hybridization detected by tyramide signal amplification.

 

A zebrafish cryosection mouse monoclonal anti–α-tubulin antibody.

 

  
Four-color fluorescence in situ hybridization on a Drosophila embryo. Bovine pulmonary artery endothelial (BPAE) cells labeled with mouse monoclonal anti–α-tubulin antibody. Reduced background staining afforded by Image-iT® FX signal enhancer.

 

  
Drosophila embryo by fluorescence in situ hybridization (FISH) with five RNA probes. A zebrafish retina cryosection visualized using TSA Kit #10. In situ hybridization of α-satellite probes to human chromosomes.

 

  
A zebrafish retina cryosection labeled with the mouse monoclonal antibody FRET 6. A zebrafish retina cryosection labeled with the mouse monoclonal antibody FRet 43.  

Advantages of Tyramide Signaling Amplification

The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label translates to practical benefits, namely ultrasensitive detection of lowabundance targets. In immuno- histochemical applications, sensitivity enhancements derived from TSA allow primary antibody dilutions to be increased in order to reduce nonspecific background signals,(6) and can overcome weak immunolabeling caused by suboptimal fixation procedures(7) or low levels of target expression.(8) The lower detection threshold of TSA compared to fluorescent secondary antibodies also allows detection of two targets with primary antibodies raised in the same host species but without substantial crosstalk between the signals.(7,9)

The increased sensitivity afforded by TSA can be critically important for detection of short oligonucleotide probes and low-abundance mRNAs by fluorescence in situ hybridization (FISH).(4,10) Optimal probe concen- trations are typically 2–10 fold lower for TSA-detected FISH than for conventional immunocytochemical detection procedures.(11)

As with other detection systems, TSA allows identification of multiple targets by simultaneously hybridized probes. Signal development using multicolored fluorescent tyramides must be carried out sequentially with a peroxidase inactivation step between each TSA reaction, to prevent crosstalk.(11)

Zenon® Antibody Labeling Kits Enhanced with TSA Technology

We have combined the ability to label and detect multiple mouse antibodies in the same sample with the ability to detect low abundance targets. For mouse IgG1 primary antibodies, we have developed Zenon® Mouse IgG1 Labeling Kits enhanced with TSA technology), which provide the necessary reagents from both the Zenon® Horseradish Peroxidase Mouse IgG1 Labeling Kit and the corresponding Alexa Fluor® TSA™ Kit, for researchers who want both the ease of labeling mouse IgG1 antibodies with Zenon® labeling reagents and the signal amplification afforded by TSA technology.

References

  1. J Immunol Methods 125:279 (1989).
  2. Cytometry 23:48 (1996).
  3. J Histochem Cytochem 45, 375 (1997).
  4. J Histochem Cytochem 47, 281 (1999).
  5. US Patent 5,731,158
  6. J Histochem Cytochem 40, 1457 (1992).
  7. J Histochem Cytochem 44, 1353 (1996).
  8. J Histochem Cytochem 45, 315 (1997).
  9. Methods 18, 459 (1999).
  10. J Histochem Cytochem 47, 431 (1999).
  11. Current Protocols in Cytometry, J.P Robinson, Ed., pp 8.9.1–8.9.16, John Wiley & Sons, (2000).
  12. J Histochem Cytochem 48, 1593 (2000).
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