Tyramide signal amplification (TSA)  

For ultimate detection sensitivity, tyramide-based amplification techniques are widely used in fluorescent immunodetection and FISH procedures to visualize targets with low expression levels or suboptimal fixation. Tyramide signal amplification (TSA) is an enzyme-mediated detection method that uses horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid in situ. This technology can be used with high-performance Alexa Fluor® dyes, conventional fluorescent dyes, or colorimetric detection systems.

Select TSA detection kits

Where is TSA used?

Immunohistochemistry

TSA for immunohistochemistryWith TSA you can detect low-abundance targets that are not detectable by conventional means. Tyramide-based signal amplification provides unprecedented sensitivity without compromising resolution.

Samples are processed with a simple incubation step, and signals can be multiplexed to explore colocalization with other amplified targets or conventional labels.

Fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH)Multiplex fluorescence in situ hybridization (FISH) enables you to assay multiple targets and visualize colocalized signals in a single specimen. Using spectrally distinct fluorophore labels for each hybridization probe, this approach gives you the power to resolve several genetic elements or multiple gene expression patterns through multicolor visual display.

When to use tyramide-based amplification

Life Technologies offers a complete range of detection technologies for fluorescence imaging. Using this suite of tools you can select the optimal technique for your target abundance with the right detection wavelength to multiplex your experiment.

  • High abundance target—primary conjugate, no amplification needed
  • Medium abundance target—secondary conjugate, modest amplification
  • Medium-low–abundance target—streptavidin conjugate, significant signal enhancement
  • Low-abundance target—enzyme amplification for maximum signal enhancement
 

New levels of fluorescence sensitivity

The signal amplification conferred by the turnover of multiple tyramide substrates for each peroxidase label enables ultrasensitive detection of low-abundance targets. With tyramide signal amplification (TSA) you can see previously undetectable targets clearly, and the range of dye colors available offers you many multiplexing options. Life Technologies offers a range of TSA kits with a choice of wavelengths for single-wavelength and multiplex detection.

 Tyramide signal amplification (TSA)Enlarge Image
Zebrafish retina with secondary immunofluorescence, TSA amplification and nucleic acid counter staining.

A zebrafish cryosection incubated with the biotin-XX conjugate of mouse monoclonal anti–a-tubulin antibody. The signal was amplified with TSA Kit #22 with HRP–streptavidin and Alexa Fluor 488 tyramide. The sample was then incubated with the mouse monoclonal FRet 6 antibody and was visualized with Alexa Fluor 647 goat anti–mouse IgG, which is pseudocolored magenta. Finally, the nuclei were counterstained with SYTOX® Orange nucleic acid stain.

Maintain resolution and reduce background

A higher dilution of primary detection reagents reduces nonspecific interactions, and TSA assays are designed to be effective with higher dilutions of antibody for extremely specific detection and low background signal. The fluorescent dye moieties deposited in the amplification reaction bind covalently to proteins close to the target, making TSA ideal for subcellular localization of proteins with high resolution.

 Tyramide signal amplification (TSA)Enlarge Image
Zebrafish retina with immune-detection, TSA amplification and nucleic acid counter staining.
A zebrafish retina cryosection was labeled with the mouse monoclonal antibody FRet 43, which was detected using TSA Kit #10 with the HRP conjugate of goat anti–mouse IgG antibody and Pacific Blue™ tyramide. The nuclei were counterstained with the SYTOX® Orange nucleic acid stain.

Use less antibody and generate more data

When your primary antibody is limiting or expensive, tyramide signal amplification (TSA) can generate sensitive signals with much higher antibody dilutions. Specificity improves because nonspecific binding is reduced at lower antibody concentrations, and you use less of your precious antibody.

 Tyramide signal amplification (TSA)Enlarge Image
Zebrafish retina with immune-detection, TSA amplification and nucleic acid counter staining.
A zebrafish retina cryosection labeled with the mouse monoclonal antibody FRet 43 and detected using TSA Kit #2 with the HRP conjugate of goat anti–mouse IgG antibody and green-fluorescent Alexa Fluor® 488 tyramide. The nuclei were counterstained with blue-fluorescent Hoechst 33258.

Easy multiplexing for colocalization

TSA allows identification of multiple targets in a sample using simultaneously hybridized probes. A simple incubation step is all that is required for each fluorescent tyramide to react with HRP and covalently link to proteins near the target. For multiplexing, the incubation steps are carried out sequentially for each fluorophore, with a peroxidase inactivation step between each TSA reaction.

 Fluorescence in situ hybridization (FISH)Enlarge image
Four-color fluorescence in situ hybridization on a Drosophila embryo. 
A late blastoderm stage (nuclear cycle 14) embryo was probed with four different RNA probes.Blue: sog labeled with DNP, followed by a rabbit anti–dinitrophenyl-KLH IgG antibody detected with an Alexa Fluor® 647 chicken anti–rabbit IgG antibody. Green: ind labeled with biotin, followed by streptavidin HRP and Alexa Fluor® 350 tyramide (TSA Kit #27. Red: msh labeled with digoxigenin followed by sheep anti-digoxigenin antibody detected with an Alexa Fluor® 488 donkey anti–sheep IgG antibody. Yellow: sna labeled with fluorescein followed by mouse anti-fluorescein antibody detected with an Alexa Fluor® 555 goat anti–mouse IgG antibodyEnlarge Image. Image contributed by Dave Kosman and Ethan Bier, University of California, San Diego.

Selection guide for TSA detection kits

Labeled tyramide
Horseradish peroxidase conjugate

Anti–mouse IgG (host: goat)

Anti–rabbit IgG (host: goat)
Streptavidin
Alexa Fluor® 350T20917T20927T20937
Alexa Fluor® 488T20912T20922T20932
Oregon Green® 488  T20939
Alexa Fluor® 546T20913T20923T20933
Alexa Fluor® 555T30953T30954T30955
Alexa Fluor® 568T20914T20924T20934
Alexa Fluor® 594T20915T20925T20935
Alexa Fluor® 647T20916T20926T20936
Biotin-XX T20911T20921T20931