Tyramide Signal Amplification (TSA)

Tyramide Signal Amplification (TSA), also known as catalyzed reporter deposition (CARD), offers an effective way to efficiently boost your signal and detection capabilities for low-abundance targets in IHC, ICC and FISH. HRP catalyzes the conversion of labeled inactive tyramide into a reactive radical, which then binds to adjacent tyrosine residues greatly enhancing the density of labeling. Tyramide reagents can be conjugated with a dye for fluorescent detection, or a hapten for antibody-based probing.

TSA can be used to enhance the signal of multiple targets on one sample by deactivating HRP using a peroxidase quenching buffer, then carrying out another round of TSA using a different set of antibodies from a different host species. Alternatively, the antibodies for the initial round of TSA can be stripped, leaving the covalently bound tyramides. TSA can then be repeated for the desired number of targets.

Figure 1: Tyramide Signal Amplification Principles: A primary and secondary antibody are used to label a tissue or cell sample. The secondary antibody is pre-conjugated to horseradish peroxidase (HRP), which in the presence of H₂O₂, catalyzes a labeled tyramide substrate into a highly reactive species that covalently bind to tyrosine residues on the proteins in close proximity to the antibodies and HRP, thus providing signal amplification.

Key Features of Tyramide Signal Amplification:

• Allows detection of low-abundance targets
• Used to enhance signals in IHC, ICC and FISH
• Reduces the amount of primary antibody required
• 100-fold more sensitive than conventional methods
• Simple, flexible, and easy to incorporate into IHC, IHC and FISH workflows
• Compatible with fluorescent multiplex systems

Featured Products: TSA Vivid™ Fluorophore Kits

Our TSA Vivid Fluorophore Kits are engineered for increased brightness and improved performance in ICC, IHC and FISH applications. They are specifically designed for optimal performance in the RNAscope™ Multiplex Fluorescent v2 Assay, enabling visualization of gene expression at the single cell level.

Key features of TSA Vivid Fluorophore Kits:

• Optimized for use with the RNAscope™ Multiplex Fluorescent v2 Assay
• Suitable for multiplexing
• Brighter than equivalent competitor fluorophores

Figures 2 and 3 demonstrate the improved brightness of TSA Vivid dyes in the RNAscope™ Multiplex Fluorescent v2 Assay versus a leading competitor.

Figure 2: 3-plex RNAscope™ Multiplex Fluorescent v2 Assay on HeLa cells with the leading competitor dyes (left) and the corresponding TSA Vivid dyes 520, 570 and 650 (right). All dyes were used at 1:1500 dilution. Markers shown are Polr2a in green, PPIB in red and UBC in white. Nuclei are counter-stained with DAPI (Cat. No. 5748).

Figure 3: 3-plex RNAscope™ Multiplex Fluorescent v2 Assay on human lung cancer tissue with the leading competitor dyes (left) and the corresponding TSA Vivid dyes 520, 570 and 650 (right). All dyes were used at 1:1500 dilution. Cancer immuno-oncology markers were probed: IDO-1 in green, KRT-19 in red and PD-1 in white. Nuclei are counter-stained with DAPI (Cat. No. 5748).

Tyramide Signal Amplification Protocol Outline for IHC and ICC

1. Follow IHC/ICC protocol for fixing, permeabilizing and blocking cells/tissue sample
2. Incubate with primary antibody
3. Incubate with HRP-conjugated secondary antibody
4. Incubate with dye-tyramide solution (e.g. Cyanine 3 Tyramide (Cat. No. 6457))
5. Visualize and/or record fluorescence intensity or repeat TSA steps as necessary to label all targets (after either quenching HRP or removing antibodies)

Tyramide Signal Amplification Product Review

Find out how our customers have used and what they think of our 5-star TSA product Biotinyl Tyramide (Cat. No. 6241), as they share experimental details and images of their results.

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