Tech Tip: Multi-Color Fluorescence Imaging Using Biotium’s TyraMax™ Amplification Dyes

Introduction

Tyramide signal amplification (TSA) is a highly sensitive method that enables the detection of low-abundance targets in immunofluorescence. The technique works through enzymatic signal amplification: after primary antibodies (or biotinylated primary antibodies) bind to their targets, an HRP-conjugated secondary antibody (or streptavidin) catalyzes the covalent coupling of labeled tyramide to tyrosine residues on nearby proteins. This enzymatic coupling reaction deposits multiple fluorescent molecules at each target site, generating significantly stronger signal than conventional labeled secondary antibodies. TSA delivers exceptional sensitivity and signal-to-background ratios, making it possible to visualize low-abundance targets that cannot be efficiently detected using traditional immunostaining methods. Additionally, TSA compensates for the reduced antibody binding efficiency inherent to formalin-fixed, paraffin embedded (FFPE) samples, improving detection sensitivity of challenging targets in highly cross-linked tissues. 

Biotium’s TyraMax™ Amplification Dyes are high-performance tyramide amplification dyes engineered to maximize the advantages of TSA while enabling robust multiplex imaging. These dyes provide bright, photostable signal with low background and exceptional chemical stability. Unlike other TSA dyes, TyraMax™ Dyes remain stable in TSA amplification buffer for up to 24 hours, making them ideal for automated staining workflows. With the widest selection of dye colors on the market, from blue to near-IR, TyraMax™ Dyes integrate seamlessly into standard multi-color panels for both conventional and spectral imaging. Together, these performance characteristics provide a reliable solution for sensitive detection and streamlined multiplexing using TSA.

In the sections below, we highlight example TSA workflows using TyraMax™ Dyes and multiplexing approaches that demonstrate how these reagents can be configured to achieve high sensitivity and flexible panel design. These workflows are not specific to TyraMax™ Dyes and can be adopted for other TSA dyes as well.

Multi-color labeling using TyraMax™ Amplification Dyes

There are three common strategies for using TSA in multi-color fluorescence imaging:

A. Use TSA in combination with dye-conjugated antibodies or other immunostaining reagents.

B. Perform multiple rounds of TSA sequentially, performing a peroxidase quenching step after each round of labeling to inactivate the secondary antibody from the previous labeling step.

C. Perform multiple rounds of TSA sequentially, performing an antigen retrieval step after each round of labeling to remove all antibodies, while the dye or biotin that is covalently attached to the sample is retained. In this way, primary antibodies from the same species can be used.

In the next three sections we will discuss each of these strategies, followed by general recommendations for multi-color imaging with TSA.

A. Using TyraMax™ Amplification Dyes in Combination with Conventional Immunostaining Methods

Our TyraMax™ Dyes enable robust, multi-color fluorescence imaging and are compatible with dye-labeled antibodies and various cell staining methods (see Figure 1). In this section we provide a sample protocol for integrating TSA with conventional immunostaining techniques.

1. Antibody and tyramide selection:
   1. Choose primary antibodies from different host species and an appropriate secondary HRP conjugate.
   2. Use a TyraMax™ Dye that can be spectrally separated by your instrument and is spectrally distinct from any other fluorescent stains that will be used. TyraMax™ Dyes are designed to be well-separated in most confocal systems. See our Tech Tip: Fluorescence Bleed-Through and Cross-Talk for more information on spectral separation for multiplexing.
2. Deparaffinization or fixation and pre-treatment:
   1. If using paraffin sections, perform deparaffinization and rehydration according to standard protocols. If necessary, perform antigen retrieval as recommended for your primary antibody. Our AntiFix™ Universal Antigen Retrieval Buffer (Cat. No. 22030) is designed for heat-induced epitope retrieval (HIER) for FFPE tissues.
   2. Fix, permeabilize, and block cell or tissue samples following general immunohistochemistry protocols.
3. Perform primary antibody labeling according to your preferred protocol and/or instructions from the primary antibody suppliers. Multiple primary antibodies from different hosts can be applied simultaneously.
4. Wash samples three times with PBS.
5. Add secondary antibodies, including an HRP-conjugated secondary antibody for TSA amplification, and incubate for 1 hour at room temperature. The HRP-mediated dye labeling reaction does not interfere with the binding of dye-labeled antibodies or other fluorescent staining reagents.
6. Perform tyramide signal amplification with the TyraMax™ Dye.
   1. Prepare working amplification buffer: 1X Tyramide Amplification Buffer Plus (Cat. No. 22029) with 0.0015% hydrogen peroxide, or 1X Ready-to-Use Tyramide Amplification Buffer (Cat. No. 22027).
   2. Prepare staining solution by diluting the tyramide solution in the working amplification buffer.
   3. Wash samples three times with PBS.
   4. Incubate samples with the staining solution for 2-10 minutes at room temperature.
7. Wash samples three times with PBS  + 0.1% Triton® X-100. Mount samples with mounting medium. For tissue samples on slides, cover with a coverslip and seal.

Figure 1. Multi-color imaging using TSA integrated into conventional immunofluorescence. (A) HeLa cells fixed with methanol; tubulin (green) detected with mouse anti-tubulin primary antibody and CF®488A Goat anti-mouse secondary antibody; mitochondria (red) visualized with rabbit anti-COXIV primary antibody and the Tyramide Amplification Kit with HRP Goat anti-rabbit IgG and CF®568 Tyramide (Cat. No. 33007); cell nuclei (blue) stained with DAPI. (B) HeLa cells fixed with formaldehyde; actin (magenta) detected with CF®640R phalloidin; Golgi bodies (green) visualized with mouse anti-Golgin-97 primary antibody and the Tyramide Amplification Kit with HRP Goat anti-mouse IgG and CF®488A Tyramide (Cat. No. 33000); cell nuclei (blue) stained with DAPI. (C) Rat brain section fixed with methanol; neuronal nuclei (green) labeled with CF®488A mouse anti-neuronal nuclei primary antibody; astrocytes (red) visualized with rabbit anti-GFAP primary antibody and Tyramide Amplification Kit with HRP Goat anti-rabbit IgG and CF®568 Tyramide (Cat. No. 33007).

B. Multiplexing with Sequential TSA – the Peroxidase Quenching Method

Multiple rounds of TSA can be performed sequentially to label different targets within a single sample using primary antibodies (including biotinylated primary antibodies) from different host species. Following primary antibody labeling, the first target is detected using an HRP-conjugated secondary antibody (or streptavidin) and a TyraMax™ Dye. A peroxidase quenching step is then required to deactivate the HRP on the secondary antibody before proceeding to another round of TSA using a different color amplification dye.

Figure 2 illustrates multi-color imaging of a tissue section sequentially labeled using this peroxidase quenching-based approach. All probes provide strong fluorescence signals with minimal background and no detectable crosstalk. In this section, we provide a protocol for performing multiplex TSA using peroxidase quenching, which can be readily combined with other cell staining methods to achieve multi-color imaging.

1. Antibody and tyramide selection:
   1. Choose primary antibodies from different host species and appropriate secondary HRP conjugates.
   2. Use TyraMax™ Dyes that can be spectrally separated by your instrument. TyraMax™ Dyes are offered as standalone dyes, as 3-color or 5-color dye sets plus DAPI counterstain, and in a sampler kit for custom panel optimization.
2. Deparaffinization or fixation and pre-treatment
   1. If using paraffin sections, perform deparaffinization and rehydration according to standard protocols. If necessary, perform antigen retrieval as recommended for your primary antibody. See our AntiFix™ Universal Antigen Retrieval Buffer (Cat. No. 22030). 
   2. Fix, permeabilize, and block cell or tissue samples following general immunohistochemistry protocols.
3. Perform primary antibody labeling according to your preferred protocol and/or instructions from the primary antibody suppliers. Multiple primary antibodies from different hosts can be applied simultaneously.
4. Wash samples three times with PBS.
5. Perform tyramide signal amplification with TyraMax™ Dye for detection of the first target.
   1. Add HRP-conjugated secondary antibody against the first target primary antibody and incubate for 1 hour at room temperature.
   2. Prepare working amplification buffer: 1X Tyramide Amplification Buffer Plus (Cat. No. 22029) with 0.0015% hydrogen peroxide, or 1X Ready-to-Use Tyramide Amplification Buffer (Cat. No. 22027).
   3. Prepare staining solution by diluting the TyraMax™ Dye in the working amplification buffer.
   4. Wash samples three times with PBS. 
   5. Incubate samples with the staining solution for 2-10 minutes at room temperature.
   6. Wash samples three times with PBS + 0.1% Triton® X-100.
6. Perform peroxidase quenching:
   1. Prepare peroxidase quenching buffer: 3% (w/w) H2O2 in PBS. We recommend preparing this buffer immediately before using. For cell samples in 96-well plates, prepare no less than 100 µL of peroxidase quenching buffer for each well. For tissue sections, make a sufficient amount of peroxidase quenching buffer so that the slides can be soaked in a container that is filled with this buffer.
   2. Quench the peroxidase activity by incubating the samples with peroxidase quenching buffer for 15 minutes at room temperature. Then, wash three times with PBS.
7. Perform a second round of protein blocking, secondary antibody staining, and tyramide signal amplification.
8. Repeat as desired for additional sets of primary and secondary antibodies.
9. Wash samples three times with PBS. Mount samples with mounting medium. For tissue samples on slides, cover with a coverslip and seal.

Figure 2. Sequential labeling using TSA with peroxidase quenching. PFA-fixed rat eye cryosection stained with Vimentin Recombinant Monoclonal Mouse Antibody (rV9) HRP conjugate with TyraMax™ 630/650 (magenta; Cat. No. 96140) followed by peroxidase quenching, GFAP Recombinant Monoclonal Mouse Antibody (rGA5) HRP conjugate with TyraMax™ 560/580 (red; Cat. No. 96139), and CF®488A PSD95 Recombinant Monoclonal Mouse Antibody (rK28/43) (green). Nuclei are stained with NucSpot® 750/780 (cyan; Cat. No. 41038).

C. Multiplexing with Sequential TSA – the Antigen Retrieval Method

Antigen retrieval–also referred to as heat-induced epitope retrieval (HIER)–is commonly used to treat paraffin-embedded tissue sections in order to expose the epitopes that become cross-linked during fixation. It also strips away all antibodies and other reagents that bind to the sample via non-covalent interactions. By performing an antigen retrieval step after tyramide labeling, all antibodies (including primary antibodies and HRP-conjugated secondary antibodies) from the previous detection round are removed (antibody stripping), leaving only the covalently bound TyraMax™ Dyes. As a result, there’s no chance of cross-reactivity even when using multiple primary antibodies from the same host species, and all targets can be visualized simultaneously after multiple rounds of TSA with different color amplification dyes.

Various devices and protocols can be used for antigen retrieval, and sometimes they need to be specially optimized for certain tissue sections and/or targets. Here, we describe a technique based on microwave treatment (MWT) that is easy, fast, and effective for most of our samples, including both paraffin-embedded and frozen tissue sections (Figure 3).

Note: Microwaving is not recommended for cells in plastic plates or dishes, which may melt when heated.

1. Antibody and tyramide selection:
   1. Primary antibodies can be from the same host species as antibodies are stripped after each round of staining.
   2. Use TyraMax™ Dyes that can be spectrally separated by your instrument. TyraMax™ Dyes are offered as standalone dyes, as 3-color or 5-color dye sets plus DAPI counterstain, and in a sampler kit for custom panel optimization.
2. Deparaffinization or fixation and pre-treatment
   1. If using paraffin sections, perform deparaffinization and rehydration according to standard protocols. If necessary, perform antigen retrieval as recommended for your primary antibody. See our AntiFix™ Universal Antigen Retrieval Buffer (Cat. No. 22030). 
   2. Fix, permeabilize, and block cell or tissue samples following general immunohistochemistry protocols.
3. Perform primary antibody labeling with the antibody against the first target according to your preferred protocol and/or instructions from the primary antibody suppliers.
4. Wash samples three times with PBS.
5. Perform tyramide signal amplification with the TyraMax™ Dye for detection of the first target.
   1. Add HRP-conjugated secondary antibody against the first target primary antibody and incubate for 1 hour at room temperature.
   2. Prepare working amplification buffer: 1X Tyramide Amplification Buffer Plus (Cat. No. 22029) with 0.0015% hydrogen peroxide, or 1X Ready-to-Use Tyramide Amplification Buffer (Cat. No. 22027).
   3. Prepare staining solution by diluting the tyramide solution in the working amplification buffer.
   4. Wash samples three times with PBS.
   5. Incubate samples with the staining solution for 2-10 minutes at room temperature.
   6. Wash samples three times with PBS + 0.1% Triton® X-100.
6. Perform antigen retrieval:
   1. Choose an antigen retrieval buffer (also known as heat induced epitope retrieval buffer, or HIER buffer) that is recommended for the primary antibody you will use for the second round of detection. The primary antibody supplier may provide information about proper antigen retrieval conditions. Biotium’s AntiFix™ Universal Antigen Retrieval Buffer (Cat. No. 22030) is designed to work well as a general HIER buffer.
   2. Place the tissue slides in a microwave-safe container and fill with the antigen retrieval buffer to cover the samples. Slides can be placed vertically or horizontally, as long as the tissue sections are fully immersed in the buffer without overflowing. We recommend using a generous amount of buffer (>50 mL) to prevent drying out during the microwave treatment. Loosely cover the container.
   3. Microwave the samples until the buffer comes to a boil. Watch closely to prevent boil-over. If it does, pause the microwave and supplement more antigen retrieval buffer. Boil for 10 minutes total.
   4. After microwave treatment, transfer the container (be careful of hot liquid) to the bench top and let it cool down to room temperature (30-60 minutes). Then, wash three times with dH2O to completely remove the antigen retrieval buffer.
7. Perform a second round of protein blocking, antibody staining, and tyramide signal amplification.
8. Repeat as desired for additional sets of primary and secondary antibodies.
9. Wash samples three times with PBS. Mount samples with mounting medium, cover with a coverslip, and seal.

Figure 3. Antigen retrieval-based sequential labeling of cytokeratin (pan-CK) and tight junctions (ZO1). (A) Paraffin-embedded human colon section sequentially labeled with two CF® Dye Tyramides: the first round with HRP Goat Anti-Mouse IgG and CF®405S Tyramide, staining cytokeratin in blue, followed by antibody removal; the second round with HRP Goat Anti-Mouse IgG and CF®568 Tyramide, staining the tight junctions in red. The nuclei were stained with NucSpot® 470 nuclear stain (Cat. No. 40083, shown in green). (B) Paraffin-embedded human colon section sequentially labeled with three CF® Dye Tyramides: the first round with HRP Goat Anti-Mouse IgG and CF®488A Tyramide staining of cytokeratin in green; the second round with HRP Goat Anti-Mouse IgG and CF®640R Tyramide staining of tight junctions in magenta; the third round with HRP Goat Anti-Mouse IgG and Cyanine 555 Tyramide staining of histone H1 in red, with antibody removal performed between each round.

Technical Tips

Among the three strategies discussed above, the antigen retrieval method offers the best signal-to-noise and the most flexible antibody selection without any issue of cross-talk. However, the microwave treatment or other antigen retrieval techniques may or may not be compatible with all applications. For example, chambered cover glass commonly used in cell imaging may be damaged during microwave treatment. Which multiplexing strategy to use should be determined experimentally based on the nature of samples and targets. The following technical tips highlight key considerations for selecting and implementing TSA-based multiplexing strategies.

• Always begin by labeling each target individually before attempting multiplexing. Troubleshoot and optimize protocols as needed, then run multiple labeling experiments after optimization is complete.
• When multiplexing, include single-stain controls for each experiment to rule out cross-talk between channels. For multi-round TSA, include controls with second round tyramide but no second round antibody to verify complete HRP stripping or inactivation after the first round of TSA.
• Make sure the sample preparation method is compatible with all targets. For instance, we found that tubulin is better detected when the samples are fixed with methanol, while formaldehyde fixation works better for actin, nucleolin, and cyclin B1 detection. Exercise caution when working with targets requiring specialized treatment.
• When working with tissue sections, evaluate batch-to-batch variations carefully. We have found that tissue sections from different suppliers or different batches from the same supplier showed dramatically different staining efficiency. We recommend running a positive control experiment for each new batch of tissue samples.
• For multi-color imaging, try to optimize the system so that labels with different colors give comparable fluorescence intensity under your microscope. For example, if the fluorescence signal in the green channel is far stronger than that of the red channel, there may be a risk of the green fluorescence bleeding into the red channel. For more information, see our Tech Tip: Fluorescence Bleed-Through and Cross-Talk.
• When performing sequential labeling using the peroxidase quenching method, it is crucial to completely quench the HRP activity before performing a subsequent round of TSA. We recommend using a generous amount of quenching buffer so that the sample is well-covered by the buffer, and the buffer should be freshly prepared. If possible, gently shake the samples during incubation.
• For sequential labeling using the peroxidase quenching method, we also recommend trying different orders of applying the antibodies and tyramides. If using one set of antibodies/tyramide followed by another set results in cross-talk (which means the target to be detected by the first set is also stained by the second set), try switching them and use the second set first. We have found that staining the less abundant target first, then staining the more abundant target, usually gives better results.
• Highly cross-adsorbed HRP-labeled secondary antibodies are recommended for tissue staining and multiplex staining using the peroxidase quenching method.

Biotium’s TSA Portfolio

Biotium offers a wide selection of reagents for TSA available as complete kits or individual components, providing the flexibility to tailor amplification strategies to specific experimental needs while maintaining consistent, high-quality performance. These reagents can be used individually or combined into complete TSA workflows, readily integrating into existing immunofluorescence protocols. Biotium’s TSA products support diverse experimental designs, from straightforward signal enhancement of a single low-abundance target to highly multiplexed panels using sequential tyramide labeling.

• TyraMax™ Dyes and Kits: Designed to yield a brighter signal compared to our original CF® Dye Tyramides. All TyraMax™ Dyes are chemically stable in amplification buffer for up to 24 hours, making them ideal for automated staining workflows with more resistance to oxidation, unlike other TSA dyes.
• CF® Dye Tyramides: Featuring our bright and photostable CF® Dyes for superior imaging performance.
• HRP-conjugated secondary antibodies: High-performance antibodies with highly cross-adsorbed options to minimize cross-reactivity.
• Tyramide Signal Amplification Kits: Kits complete with CF® Dye Tyramides or biotin-XX-tyramide, HRP-conjugated secondary antibodies or streptavidin, and optimized buffers.
• Tyramide Amplification Buffer Plus: Our enhanced buffer formulation, delivering improved brightness, specificity, and sensitivity over our original buffer below.
• Ready-to-Use Tyramide Amplification Buffer: Our original buffer with a convenient formulation requiring no addition of hydrogen peroxide.
• AntiFix™ Universal Antigen Retrieval Buffer: Designed for heat-induced epitope retrieval (HIER) of FFPE tissues and validated in TSA applications with our CF® Dyes.

Tyramide Signal Amplification Kits

Standalone Dye and Hapten Labeled Tyramides

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