Questions and Answers About Tyramide Signal Amplification (TSA)

Questions & Answers

1. What is Tyramide Signal Amplification (TSA™)?
Tyramide signal amplification is a novel technology developed and patented by PerkinElmer scientists. It is an extremely powerful signal amplification technology that can be used in many applications including immunohistochemistry, in situ hybridization, electron microscopy, ELISA, and others. It can be used with both chromogenic and fluorescence visualization methods. Additional advantages of the tremendous boost in signal from the TSA reagents include the potential to reduce the use of valuable reagents or improve signal to noise by reducing the titer of a problematic reagent in the assay protocol. Use of TSA facilitates multi-target detection in both IHC and (F)ISH applications. The TSA reagents can be easily added to any standard IHC protocol. For (F)ISH applications, standard nonradioactive DNA, RNA, and oligo probe labeling procedures can be used for probe generation, using common nonradioactive labeling haptens such as biotin, fluorescein, and digoxigenin. After standard hybridization protocols, immunological detection steps can be easily modified to include TSA.

2. What is tyramide?
Tyramide is the amplification reagent. It is a phenolic compound that, when activated by the enzyme horseradish peroxidase (HRP), covalently binds to electron rich moieties on a surface (i.e., predominantly to tyrosine residues in proteins in tissue or cell preparations).

3. How does the signal amplification work?
Tyramide Signal Amplification is based upon the patented catalyzed reporter deposition (CARD) technique using derivatized tyramide. In the presence of small amounts of hydrogen peroxide, immobilized HRP converts the labeled substrate (tyramide) into a short-lived, extremely reactive intermediate. The activated substrate molecules then very rapidly react with and covalently bind to electron rich regions of adjacent proteins. This binding of the activated tyramide molecules occurs only immediately adjacent to the sites at which the activating HRP enzyme is bound. Multiple deposition of the labeled tyramide occurs in a very short time (generally within 3-10 minutes). Subsequent detection of the label yields an effectively large amplification of signal.

4. What is "TSA Plus DNP"?
In the TSA Plus DNP (HRP or AP) System, the amplification reagent is labeled with 2,4-dinitrophenyl (DNP). Similar to the commonly used digoxigenin hapten label, DNP is a small molecule that rarely occurs in the natural world. Antibodies to the DNP hapten label are thus very unlikely to bind nonspecifically to other biological material. The ""Plus"" refers to proprietary changes made to the amplification diluent which provide enhanced sensitivity.

5. Do I have to change my standard protocol in order to add TSA™ to my protocol?
Slides to be detected with TSA enhancement should be prepared using standard fixation and/or embedding techniques. All common labeling haptens- biotin, fluorescein, or digoxigenin- are compatible for use with TSA. The only protocol requirement for adding TSA is the inclusion of an HRP reagent prior to the addition of the amplification reagent. It is important however, to titer reagents such as primary antibody concentration, enzyme conjugate concentration, etc., in order to achieve maximum signal enhancement.

6. Can I always use TSA™ in my ISH or IHC applications?
TSA™ is a signal amplification system catalyzed by HRP. As long as HRP is or can be an integral part of the detection protocol, TSA can be applied. The only requirement is that an HRP conjugate must be included in the protocol immediately prior to the addition of the amplification reagent. Streptavidin-HRP is included in the TSA kits for use with biotin-labeled antibodies or probes. Other hapten/antibody or probe combinations can be used as well- for example, biotin/ABC, fluorescein/anti-fluorescein-HRP, digoxigenin/ anti-digoxigenin-HRP, etc.

7. Does TSA™ work on all ISH and IHC sample types?
TSA™ has been successfully applied to tissue sections, cultured cells (animal and bacterial) plant tissue, invertebrate embryos, and chromosome spreads.

8. Is TSA™ compatible with my fixation or embedding procedure?
TSA™ has been successfully applied to formalin-fixed/paraffin-embedded sections, paraformaldehyde-fixed cultured cells, frozen sections, various EM embedding materials, and some plastic-embedded sections (e.g. methyl methacrylate).

9. Is TSA™ more sensitive than all other standard fluorescence and chromogenic detection systems, including using Avidin Biotin Complex (ABC) reagents?
YES. TSA is significantly more sensitive compared to ANY standard system currently available, including ABC. The ABC reagents can be used instead of streptavidin-HRP, before and/or after the amplification reagent.

10. How much time will TSA™ add to my protocol?
Addition of TSA adds very little time to standard IHC and ISH protocols. In most protocols the amplification step can be complete in 3 to 10 minutes. Both the TSA Biotin and TSA Plus DNP Systems add about 40 minutes additional reagent incubation time: 3-10 minutes for the labeled tyramide plus 30 minutes for the enzyme conjugate. The TSA Fluorescence Systems add even less time as the amplification reagent is directly labeled with a fluorophore molecule. The fluorophore tyramide is incubated for 3-10 minutes, after which signal can be directly visualized with a fluorescence microscope. 11. Does TSA™ decrease resolution?
TSA should not lead to a noticeable decrease in resolution. Titration of assay reagents and/or decrease in amplification reaction time can improve spatial resolution if needed.

12. Does TSA™ increase my background?
Background can be caused by a number of factors. Some are inherent to the tissues/cells being stained such as high endogenous enzyme activity or high endogenous biotin. Nonspecific binding of reagents, such as second step antibodies, or technique problems, such as absence of a blocking step or poor/inadequate washing, can also contribute to high background. The TSA reagents themselves do not lead to significant increase in background staining provided that reagents used have been properly titrated. However, as the activation of the amplification reagent is catalyzed by HRP, it is imperative to quench any endogenous peroxidase activity prior to adding the amplification reagent to the slide. The kit manuals include recommended procedures for the quenching of endogenous peroxidase activity, as well as potential solutions to other background problems encountered. Control slides are highly recommended, especially to determine sources of background staining and efficacy of background control measures. Use of TSA can actually improve signal to noise. In some staining applications secondary antibody reagents must be used at fairly high concentrations in order to obtain any visualization of the desired signal. Because of its powerful signal enhancement, adding TSA to this protocol can allow this problematic reagent to be used at a more dilute concentration. Signal will remain but the background can be significantly reduced or eliminated.

13. How does the TSA™ technology compare to IS-PCR?
Several publications have cited the TSA™ products as an alternative to in situ PCR particularly for detection of single copy viral nucleic acid. TSA is simpler to learn and use, requires the use of no special or expensive equipment and fewer control slides are needed to validate results. More importantly, reproducibility and retention of sample morphology are much better in samples processed with TSA as opposed to IS-PCR. Additionally, artifacts or back-diffusion of amplicons can contribute to the generation of false positive results using the IS-PCR technique. In the conclusions of a recently published paper (K. H. Wiedorn et al. 1999. Comparison of in situ hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification in the detection of HPV. Histochem. Cell Biol. 111:89-95), the authors stated " We conclude that with regard to sensitivity, for many applications the use of ISH in combination with TSA is a good alternative to the cumbersome and often poorly reproducible IS-PCR due to the high signal amplification power of TSA. Concerning reproducibility and reliability the system based on in situ signal amplification by TSA is much better than that based on target amplification by indirect IS-PCR and, therefore, represents the technique of choice."

14. Can I use TSA™ for electron microscopy applications?
Yes. Several publications have demonstrated the successful use of TSA for both IHC and ISH at the EM level. Several different approaches were used such as pre-embedding labeling followed by post-embedding detection, visualization with DAB, and visualization via the use of ultrasmall or colloidal gold reagents.

15. Can I use TSA™ in FACS?
TSA has been successfully added to FACS protocols in which the cells are fixed prior to staining. To date, no protocols are available for adding TSA to FACS protocols using live, unfixed cells.

16. Can I use TSA™ in blotting applications?
The TSA kits are designed for use in slide-based applications. For blotting applications, the PerkinElmer BLAST® kit (BLotting Amplification SysTem) provides reagents optimized for use in the amplification of signal in chromogenic Western blotting applications. The BLAST reagents are easily inserted into a standard chromogenic Western blotting protocol resulting in an 8 to 10-fold amplification of signal. BLAST uses immobilized HRP to catalyze the deposition of the biotin-labeled amplification reagent onto membranes that have been previously blocked with protein. The deposited biotin labels can then be subsequently detected using Streptavidin-HRP followed by 4CN Plus chromogenic substrate. The kits provide sufficient reagents for use for 500cm2 (Cat.# NEL761A) or 2500 cm2 (Cat.# NEL761) of membrane. Kit components include biotin-labeled amplification reagent, amplification diluent, streptavidin-HRP, 4CN Plus chromogenic substrate and diluent, control protein, and a blocking reagent.

17. Can I use TSA™ in enzyme immunoassays?
The TSA kits are designed for use in slide-based applications. For ELISA applications, the PerkinElmer ELAST™ kit (ELISA Amplification SysTem) provides reagents optimized for use in the amplification of solution phase signal in microplate-based assays. The ELAST reagents are easily inserted into standard chromogenic ELISA protocols, resulting in an 8 to 32-fold improvement of sensitivity. ELAST uses immobilized HRP to catalyze the deposition of the biotin-labeled amplification reagent onto the solid phase, which has been previously blocked with protein. The deposited biotin labels can then be subsequently detected using Streptavidin-enzyme conjugate, followed by the appropriate solution phase chromogen such as OPD or TMB. The kit (Cat.#NEP116) provides reagents sufficient for amplification of signal in ten 96-well microplates. Kit components include the biotin-labeled tyramide amplification reagent, 2X amplification diluent, and streptavidin-HRP concentrate.

18. Can I do multi-target/ multi-color detection?
Sequential multi-target/ multi-color detection is possible. Multi-target detection has successfully been done with TSA in IHC applications in which the two primary antibodies were derived from different or even from the same species. Multi-target detection using TSA in (F)ISH applications has been shown in a variety of formats: one target radiometric probe/ second target nonrad probe with chromogenic detection, both targets with nonrad probes with chromogenic detection, two or more targets with nonrad probes with fluorescence signal output. TSA has also facilitated the detection of both nucleic acid and protein targets on the same slide.

19. What is included in the kit?
TSA Biotin System: Biotin-labeled amplification reagent, 1X Amplification diluent, Blocking reagent, and Streptavidin-HRP
TSA Fluorescence Systems: Fluorophore-labeled amplification reagent, 1X Amplification diluent, Blocking reagent, and Streptavidin-HRP. Fluorophore labels available include fluorescein, tetramethylrhodamine, coumarin, Cyanine 3, and Cyanine 5.
TSA Plus DNP (HRP or AP) System: DNP-labeled amplification reagent, 1X Plus Amplification reagent, Blocking reagent and EITHER anti-DNP-HRP OR anti-DNP-Alkaline phosphatase.

20. What kit sizes are available?
TSA™ kits come in a number of sizes. The number of slides that can be stained depends on the amount of reagent required to completely cover the tissue section or cells being stained. The indicated number of slides per kit is based on using the amplification reagent in a volume of 100-300uL. The TSA Product Listing provides a detailed list of kit sizes available.

21. Are there specific kits available for ISH and IHC?
No. Reagents supplied in the TSA kit products can be used for both IHC and (F)ISH applications. Instructions are provided in the product insert for both applications.

22. What options do I have for visualization of my signal?
The TSA Biotin System provides the option of either indirect chromogenic or fluorescence signal output. The biotin-labeled amplification reagent is covalently deposited through the action of immobilized HRP enzyme. For chromogenic detection, the biotin labels can subsequently be detected with a second enzyme step followed by the appropriate chromogen-e.g. SA-HRP followed by DAB, SA-AP followed by BCIP/NBT. Alternatively for fluorescence signal, the biotinyl tyramide amplification reagent can be followed by a SA-fluorophore conjugate. PerkinElmer offers a choice of three: SA- Fluorescein, SA-Texas Red®, and SA-Coumarin.

In the TSA Fluorescence System kits, the amplification reagent is labeled with a choice of five different fluorophores- fluorescein, tetramethylrhodamine, coumarin, Cyanine 3, and Cyanine 5. The TSA Fluorescence System kits give a detectable signal with the amplification- i.e.; the deposited fluorophore labels can be immediately visualized after the amplification step. If desired, the TSA Fluorescence System-Fluorescein kit can be used to provide a chromogenic signal. The fluorescein tyramide amplification step can be followed by an anti-fluorescein enzyme conjugate and then visualized with the appropriate chromogen.

Signal is visualized using the TSA Plus DNP (HRP or AP) System by indirect chromogenic detection. The DNP-labeled amplification reagent is covalently deposited through the action of immobilized HRP enzyme. The DNP labels can then be detected using either anti-DNP-HRP or anti-DNP-alkaline phosphatase (supplied in kit), followed by an appropriate chromogen.

23. What are the excitation and emission values for the fluorophores used in the TSA Fluorescence System kits and complementary products?

Cyanine 3550nm570nm
Cyanine 5648nm667nm
Texas Red™593nm612nm

24. What chromogenic substrates are compatible with TSA™?
TSA kits have been proven to work with any standard chromogenic substrates such as DAB (with or without Nickel enhancement), AEC, BCIP/NBT, Fast Red, etc.

25. Due to the loss of antigenicity in formalin fixed, paraffin-embedded sections, I am forced to use my antibody on frozen (cryostat) sections. Can TSA™ help?
Antigen retrieval techniques, usually involving the application of heat through boiling or microwaving, can be used to attempt to revive antigenicity of target proteins in the paraffin-embedded sections. With the powerful enhancement of signal achieved through the use of TSA, signal may be detected in these types of sections without the need to add antigen retrieval. However, in some instance, optimal results will be achieved through the addition of both techniques to the standard IHC protocol.

26. Can I do multiple cycles of amplification if one cycle does not give me the sensitivity I need?
Yes. After the labeled tyramide amplification reagent is deposited, the appropriate enzyme conjugate (SA-HRP, anti-fluorescein-HRP, or anti-DNP-HRP) is re-applied. This is followed by a second application of the amplification reagent followed by the standard visualization reagents. Several research reports have indicated that more than three rounds of amplification may lead to loss of signal resolution.

27. What type of control slides should I use with TSA™?
Control slides help to prove the scientific validity of experimental results and should be included in every experiment. In addition, control slides can also be helpful in determining sources of nonspecific background staining. It is recommended that both an unamplified control and amplified negative control slide be run with each experiment. For ISH applications the unamplified control slide includes specific probe and all detection reagents except the TSA reagents. The amplified negative control slide should be hybridized with either no probe or a nonspecific probe and then with all detection reagents including TSA. In IHC applications the unamplified control slide should include specific primary antibody and all detection reagents except the TSA reagents. The amplified negative control slide is prepared by eliminating the primary antibody or using a nonspecific antibody, followed by all of the detection reagents including TSA.

28. Can different blocking reagents be used?
The PerkinElmer Blocking Reagent supplied in the kit has been used at PerkinElmer to qualify the kit reagents. Literature data shows that alternatives such as normal sera, BSA, casein, and others can also be used. Alternative reagents should be validated in your own application.

29. My tissue contains high endogenous biotin levels. Can TSA™ be used?
Yes. The TSA Plus DNP (HRP or AP) System is ideal for use with tissues in which endogenous biotin is problematic. The DNP-labeled amplification reagent provides a completely biotin-free method of signal enhancement.

30. How do I control endogenous peroxidase activity?
Activation and covalent binding of the labeled Amplification Reagent is catalyzed by HRP. To minimize background, endogenous peroxidase activity, if present, must be quenched prior to the addition of the TSA amplification reagent. Options include incubation of the slides in 0.3% to 3% H2O2 for 10 to 60 minutes. Methanol or PBS may be used as the diluent for H2O2. For paraffin-embedded tissues, quenching can be done after dewaxing and alcohol rehydration but before the protease digestion step. For frozen tissue or cell preps, quenching can be done following fixation and before the protease digestion step. Users should establish the need for and optimal methodology specific to the tissues or cells being stained.

31. I work with cells which autofluoresce. Can I use TSA™?
Autofluorescence is a problem in the visible light range. Use of the TSA Fluorescence System Cyanine 5 kit may allow better discrimination of specific signal, as autofluorescence is not visible in the far-red region. Cyanine 5 emits light in the far-red region (650-725nm).

32. What do I need in order to visualize Cyanine 5?
Cyanine 5 emits light in the light range approaching the far-red region (excitation 650nm/ emission 725nm). You will need an appropriate filter for your fluorescence microscope. In addition visualization of Cyanine 5 requires the use of digital imaging-.i.e. through the use of a CCD camera (cooled or uncooled) with your conventional or confocal fluorescence microscope.

33. How stable are the TSA kits?
PerkinElmer guarantees stability of 6 months after purchase for the TSA Biotin and TSA Plus DNP (HRP or AP) System kits. The TSA Fluorescence System kits are guaranteed for 3 months stability after purchase.

34. How are the reagents shipped and how should they be stored?
The TSA kits are shipped on blue ice. They should be stored at 4C upon arrival. The amplification is supplied in dried form and may not be visible in the vial. This reagent must be reconstituted using high quality DMSO or distilled, deionized water according to the kit instructions. Once reconstituted, the tyramide stock solutions should also be stored at 4C. Note: DMSO freezes at 4C. Allow stock solutions prepared with DMSO to thaw prior to use.

35. Can I buy the amplification reagent alone?
Yes. TSA individual reagent packs are available for the biotin and fluorophore-labeled amplification reagents. Each reagent pack consists of the labeled amplification reagent and 1X amplification diluent. Researchers must supply all other needed reagents including HRP conjugate and blocking reagent. First time TSA users are strongly urged to use the complete kit product, as the kit includes reagents that have been pre-optimized to work together along with detailed protocol suggestions. For large volume users, TSA Individual reagent packs are available in a 5X size. See the TSA Product List for further details.

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