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Tyramide signal amplification for low-abundance targets
Invitrogen SuperBoost tyramide signal amplification kits and reagents are specifically designed for exceptional signal amplification, offering additional signal definition and clarity required for imaging of low-abundance targets. Combining the brightness of Invitrogen Alexa Fluor dyes with poly-HRP–mediated tyramide signal amplification, the SuperBoost reagent generates sensitivity typically 2 to 10 times above that of standard treatments and other tyramide signal amplification reagents. This is particularly useful for immunohistochemistry, FISH, and other multiplexing immunophenotyping methods.
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How does tyramide signal amplification work?
Tyramide signal amplification process includes the use of horse radish peroxidase (HRP) to enzymatically convert fluorophore tyramides to bind tyrosine residues on and surrounding the protein epitope targeted by the primary antibody. As a controlled enzymatic reaction, tyramide signal amplification does not diffuse from the site of enzyme activity and therefore, provides better spatial resolution as compared to HRP or alkaline phosphatase-based methods.
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Figure 1. Illustration of the SuperBoost tyramide signal amplification system. The antigen is detected by a primary antibody (blue), followed by a poly–horseradish peroxidase (poly-HRP) conjugated secondary antibody (yellow). Activation of the dye-labeled tyramide (green) by HRP results in localized deposition of the activated tyramide derivative (pink).
What is SuperBoost tyramide signal amplification?
A limitation to IHC, spatial proteomics, and other multiplexing imaging experiments is detecting low-abundance protein and autofluorescence for non-specific binding. SuperBoost Tyramide kits and reagents use poly-HRP–conjugated secondary antibodies as a method of amplification. Antibodies with multiple HRPs confer enhanced signal without losing tissue penetration.
Benefits include:
- A highly sensitive fluorescent imaging detection method for low-abundance, hard-to-detect targets
- Easy-to-use kits that produce signals compatible with standard filters and microscopes
- High-resolution images and multiplex compatibility with fluorescent proteins, DAPI, secondary antibodies, and other SuperBoost kits
Figure 2. SuperBoost Tyramide kits and reagents with poly-HRP show brighter signal for longer duration. HeLa cells were incubated with various concentrations of anti-prohibitin antibody (manufacturer recommends a 1:150 dilution, or 5 µg/mL final), then labeled with the reagents in (1) the Invitrogen Alexa Fluor 488 Tyramide SuperBoost Kit (goat anti–rabbit IgG and Alexa Fluor 488 Tyramide); (2) our original Invitrogen TSA Kit #12 (goat anti–rabbit IgG and Alexa Fluor 488 Tyramide); or (3) an Invitrogen F(ab′)2 rabbit anti–goat IgG (H+L) secondary antibody. Cell images were captured from each treatment (using the same exposure and gain) with an Invitrogen EVOS FL Auto Imaging System (see information about EVOS imaging systems). These images indicate that the Alexa Fluor 488 Tyramide SuperBoost Kit offers higher-sensitivity detection than either our original TSA kits or directly labeled secondary antibodies.
How to use SuperBoost tyramide signal amplification
SuperBoost tyramide signal kits are simple to use and incorporate by itself or with other reagents and fluorophores. In this workflow, the fluorophore-conjugated secondary antibodies are replaced with SuperBoost tyramide signal amplification including secondary antibodies conjugated with poly-HRP. The only additional steps are incubation with conjugated tyramides for 2–10 minutes and addition of stop solution to halt HRP activity once the specific signal is detected. The stop solution additionally helps maintain the specificity and resolution of fluorescent signal. SuperBoost tyramide signal amplification kits offer a simple workflow like those used in standard ICC, IHC, and FISH.
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Figure 3. Workflow for SuperBoost tyramide signal kits. This is a six- or seven-step process that can be optimized to provide clear and bright signal. This workflow indicates where to add other fluorescent reagents and secondary antibody fluorophores.
Detecting protein expression depends on the primary clone and secondary detection method. SuperBoost tyramide signal should be used to amplify signal identifying low abundance proteins. We offer a range of detection technologies that help detect different protein abundance.
- 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.
View the immunofluorescence guide to choose appropriate detection technologies.
Examples of SuperBoost tyramide based amplification in IF, IHC, and FISH
SuperBoost tyramide signal kits are compatible with a range of other marker detection and cell staining techniques, enabling multiplex experiments and fluorescence colocalization studies. SuperBoost tyramide signal kits work with cell types and fluorescence imaging systems commonly used in standard ICC, IHC, and FISH methods. We have tested the performance of SuperBoost tyramide signal kits using formaldehyde-fixed cell lines in 2D and 3D cultures, FFPE tissues, and cryosectioned tissues.
SuperBoost tyramide signal reagent multiplexing can be achieved with:
- Fluorescent markers for counterstaining, such as DAPI
- Fluorescent proteins (i.e., GFP & RFP)
- Standard ICC/IHC
- Other SuperBoost tyramide signal kits
With IHC and FFPE samples
Sample type: Rat intestinal section (FFPE).
Antibodies: Immunolabeled sequentially with three primary antibodies against H2B, actin and Ki-67.
Method: In between each antibody labeling, samples were microwaved in citrate buffer pH6 on high until boiling (~2 min), then microwaved for 15 minutes at 20% power and then allowed to cool to room temperature before labeling with the next rabbit antibody. Samples were then labeled with 3 different primary antibodies: anti-H2B detected with Alexa Fluor 647 Tyramide SuperBoost Kit (green), rabbit anti–smooth muscle actin antibody (detected with the Alexa Fluor 488 Tyramide SuperBoost Kit (red), and rabbit anti-Ki67 antibody (detected with the Alexa Fluor 594 Tyramide SuperBoost Kit (blue).
With ICC in cell culture
Sample type: Fixed and permeabilized HeLa cells.
Antibodies: Immunolabeled sequentially with three primary antibodies against anti–ATP synthase antibody and anti–β-catenin antibody.
Method: Cells were labeled with anti–ATP synthase antibody and an Alexa Fluor 594–conjugated secondary antibody. Additionally, the cells were incubated with an anti–β-catenin antibody and labeled with the reagents in the Alexa Fluor 488 Tyramide SuperBoost Kit (goat anti–mouse IgG and Alexa Fluor 488 tyramide). Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired on a confocal microscope.
With FISH and cell culture
Sample type: Fixed and permeabilized U2OS cells.
Antibodies: Immunolabled with anti-Cas9 antibody and probed with an oligo targeting the hprt gene.
Method: U2OS cells were fixed and permeabilized and then incubated with an hprt gene probes plus inactive Cas9 protein. Hprt probes were designed for Cas9 recognition, containing sg-RNA. To detect Cas9 protein and hprt probe complex assembled at hprt loci, anti-Cas9 antibody was used. This primary antibody was detected by Alexa Fluor 488 Tyramide SuperBoost Kit (goat anti–mouse IgG and Alexa Fluor 488 tyramide) detecting hprt loci specifically. Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired and analyzed on an EVOS FL Auto Imaging System (see information about EVOS imaging systems).
With standard ICC/IHC
Sample type: Cultured HeLa cells, fixed and permeabilized.
Antibodies: Cells were immunolabeled with an anti–ATP synthase subunit IF1 antibody.
Method: Fixed and permeabilized HeLa cells, treated using the reagents in the Image-iT Fixation/Permeabilization Kit, were incubated with an anti-tubulin primary antibody and an Alexa Fluor 488 goat anti–mouse IgG (H+L) secondary antibody. Cells were then incubated with an anti–ATP synthase subunit IF1 antibody and labeled with the reagents in the Alexa Fluor 594 Tyramide SuperBoost Kit (goat anti–mouse IgG and Alexa Fluor 594 tyramide). Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired on a confocal microscope.
With another SuperBoost Kit
Sample type: Cultured HeLa cells, fixed and permeabilized.
Antibodies: HeLa cells were immunolabeled with an anti-prohibitin antibody.
Method: Fixed and permeabilized HeLa cells, treated using the reagents in the Image-iT Fixation/Permeabilization Kit, were incubated with an anti-prohibitin antibody and labeled with the reagents in the Alexa Fluor 647 Tyramide SuperBoost Kit (goat anti–rabbit IgG and Alexa Fluor 647 tyramide). Additionally, cells were incubated with anti–β-catenin and labeled with the reagents in the Alexa Fluor 488 Tyramide SuperBoost Kit (goat anti–mouse IgG and Alexa Fluor 488 tyramide). Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired on a confocal microscope.
Ordering information
Ordering information for Tyramide SuperBoost Kits
| Labeled tyramide (Ex/Em) |
Tyramide SuperBoost Kits*
| ||
|---|---|---|---|
Anti–mouse IgG (host: goat)
|
Anti–rabbit IgG (host: goat)
|
Streptavidin
| |
| Alexa Fluor 488 (495/519 nm) | B40912 B40941 (50 coverslips) | B40922 B40943 (50 coverslips) | B40932 |
| Alexa Fluor 555 (555/565 nm) | B40913 | B40923 | B40933 |
| Alexa Fluor 594 (591/617 nm) | B40915 B40942 (50 coverslips) | B40925 B40944 (50 coverslips) | B40935 |
| Alexa Fluor 647 (650/668 nm) | B40916 | B40926 | B40936 |
| Biotin-XX | B40911 | B40921 | B40931 |
| * Unless otherwise stated, sufficient material is provided for up to 150 18 mm x 18 mm coverslips (if using 150 µL in most critical incubation steps). Volumes can be adjusted for samples of different sizes. | |||
Ordering information for stand-alone Tyramide SuperBoost reagents
SlowFade antifade mountant ordering information
Use SlowFade antifade mountant when stripping and reprobing with TSA reagents.
Protocol for SuperBoost tyramide signal and applications including stripping and restaining
Featured resources
Selected publications with detailed use
- Avens HJ, Berron BJ, May AM, Voigt KR, Seedorf GJ, Balasubramaniam V, Bowman CN. Sensitive immunofluorescent staining of cells via generation of fluorescent nanoscale polymer films in response to biorecognition. J Histochem Cytochem. 2011 Jan;59(1):76-87. PMID: 21339175 .
- Kosmac K, Peck BD, Walton RG, Mula J, Kern PA, Bamman MM, Dennis RA, Jacobs CA, Lattermann C, Johnson DL, Peterson CA. Immunohistochemical Identification of Human Skeletal Muscle Macrophages. Bio Protoc. 2018 Jun 20;8(12):e2883. PMID: 30148186 .
- Tóth ZE, Mezey E. Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J Histochem Cytochem. 2007 Jun;55(6):545-54. PMID: 17242468 .
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