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Tyramide Signal Amplification and Deposition |
Tyramide signal amplification is a technique that improves the detection of low abundance targets in imaging by covalently depositing fluorescent or chromogenic reporter molecules onto the target. Invitrogen Alexa Fluor Tyramide SuperBoost kits provide exceptional signal amplification and clarity, while SuperBoost colorimetric kits with EverRed and EverBlue allow for permanent staining in colorimetric immunohistochemistry (IHC) experiments.
What is tyramide signal amplification?
Tyramide signal amplification is a technique used to enhance the detection of low-abundance targets in imaging applications based on the enzymatic deposition of fluorescent or chromogenic reporter molecules onto the target of interest. Alexa Fluor Tyramide SuperBoost kits and reagents are specifically designed for exceptional signal amplification, offering additional signal definition and clarity required for fluorescent imaging of low-abundance targets. SuperBoost EverRed and EverBlue colorimetric kits and reagents can also be used for permanent staining in colorimetric IHC experiments for multiplexing with DAB substrate or as an alternative colorimetric stain.
How does tyramide signal amplification work?
Tyramide signal amplification used in the SuperBoost kits utilizes the catalytic activity of horseradish peroxidase (HRP) for high-density labeling of a target protein or nucleic acid sequence in situ. Typical immunocytochemistry (ICC), immunohistochemistry (IHC) and in situ hybridization (ISH) experiments using the SuperBoost tyramide reagents require 10–5000 times less primary antibody than standard ICC/IHC/ISH experiments to achieve the same signal intensity. Since the SuperBoost kits greatly enhance specific signal intensity over background, they can be easily optimized to detect specific signal in samples where high endogenous autofluorescence is observed.
The tyramide signal amplification process includes the use of horseradish peroxidase (HRP) to enzymatically convert fluorophore or chromogen tyramides to covalently 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.
Click image to enlarge
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).
Benefits of tyramide signal amplification and deposition
- Enable excellent detection of low-abundance targets due to poly HRP secondary antibody
- Ability to label with antibodies from the same species due to covalent attachment of the fluorophore, allowing antibody stripping in between rounds of tyramide signal amplification
- High sensitivity allows the use of significantly lower concentrations of primary antibody
Table 1. Invitrogen tyramide signal amplification and deposition reagent options
| Alexa Fluor Tyramide SuperBoost Reagents | SuperBoost EverRed and EverBlue Tyramide Reagents | |
|---|---|---|
| Detection | Fluorescence | Colorimetric and fluorescence |
| Use | Single or 2-plex staining for fluorescence ICC, IHC, and ISH | Single or multiplex permanent colorimetric staining for IHC |
| Brightness | Higher | High |
| Sample types | Cells or Tissues | Tissues |
Alexa Fluor Tyramide SuperBoost kits for signal amplification for ICC/IHC
Combining the brightness of Invitrogen Alexa Fluor dyes with poly-HRP–mediated tyramide signal amplification, SuperBoost reagents yield precision and sensitivity 10–200 times greater than standard ICC/ IHC/ISH and 2–10 times that of other tyramide amplification techniques.
Benefits of the SuperBoost tyramide signal amplification kits 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 does SuperBoost tyramide signal amplification work?
Excellent for 1 or 2 plex imaging, Alexa Fluor Tyramide SuperBoost 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.
Click image to enlarge
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.
Examples of SuperBoost tyramide based amplification in IF, IHC, and ISH
Alexa Fluor Tyramide SuperBoost kits are compatible with a range of other marker detection and cell staining techniques, enabling multiplex experiments and fluorescence colocalization studies. These kits work with cell types and fluorescence imaging systems commonly used in standard ICC, IHC, and ISH 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
- Additional Alexa Fluor Tyramide SuperBoost kits
Table 2. Alexa Fluor Tyramide SuperBoost reagent selection guide
| Alexa Fluor 350 Tyramide Reagent | Alexa Fluor 488 Tyramide Reagent | Alexa Fluor 546 Tyramide Reagent | Alexa Fluor 555 Tyramide Reagent | Alexa Fluor 568 Tyramide Reagent | Alexa Fluor 594 Tyramide Reagent | Alexa Fluor 647 Tyramide Reagent | Alexa Fluor Plus 750 Tyramide Reagent | Biotin-XX | |
|---|---|---|---|---|---|---|---|---|---|
| Detection | Fluorescence | ||||||||
| Ex/Em | 347/442 | 495/519 | 556/573 | 555/565 | 579/604 | 591/617 | 650/668 | 750/790 | N/A |
| Channel | 430 | 488 | 514 | 555 | 594 | 594 | 647 | 750 | N/A |
| EVOS filter | CFP | GFP | YFP | RFP | Texas Red | Texas Red | Cy5 | Cy7 | N/A |
| Application | Single or 2-plex staining for fluorescence ICC, IHC, and ISH | ||||||||
| Key benefits |
| ||||||||
| Tyramide Reagent | B40952 | B40953 | B40954 | B40955 | B40956 | B40957 | B40958 | B56131 | B40951 |
| SuperBoost Kits: goat anti-mouse | B40912 B40941 (50 coverslips) | B40913 | B40915 B40942 (50 coverslips) | B40916 | B40911 | ||||
| SuperBoost Kits: goat anti-rabbit | B40922 B40943 (50 coverslips) | B40923 | B40925 B40944 (50 coverslips) | B40926 | B40921 | ||||
| SuperBoost kits: Streptavidin | B40932 | B40933 | B40935 | B40921 | B40931 | ||||
Table 3. Ordering information for stand-alone poly HRP antibodies
Alexa Fluor Tyramide SuperBoost experimental results
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 labeled with 3 different primary antibodies: rabbit anti-H2B detected with Alexa Fluor 647 Tyramide SuperBoost Kit (green), rabbit anti–smooth muscle actin antibody detected with Alexa Fluor 488 Tyramide SuperBoost Kit (red), and rabbit anti-Ki67 antibody detected with Alexa Fluor 594 Tyramide SuperBoost Kit (blue).
With ICC in cell culture
Sample type: Fixed and permeabilized HeLa cells.
Antibodies: Immunolabeled sequentially with 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–tubulin and 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 and an anti–β-catenin 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.
SuperBoost EverRed and EverBlue kits for permanent colorimetric IHC
SuperBoost colorimetric signal anchoring, achieved with SuperBoost EverRed and EverBlue Colorimetric HRP Kits, enables highly sensitive and permanent target detection in multiplexable colorimetric IHC experiments. It combines poly-HRP-mediated tyramide signal deposition with SuperBoost EverRed and EverBlue for precise and sensitive colorimetric tissue staining. These SuperBoost kits use HRP catalytic activity for high-density labeling, resulting in wash-resistant, mountable, and archivable staining. SuperBoost EverRed and EverBlue provide sharp and intense signals without the need for air-drying or unconventional protocols. These user-friendly kits work with various tissue types and can be imaged with any brightfield microscope, yielding high-resolution images.
Benefits of the SuperBoost EverRed and EverBlue HRP Kits include:
- Permanent colorimetric staining that is also fluorescent, allowing confirmation of the staining pattern with both brightfield and fluorescence imaging
- Compatibility with other common colorimetric counterstains
- Multiplexable between EverRed, EverBlue, and DAB
How does SuperBoost color signal anchoring work?
SuperBoost EverRed and EverBlue HRP Kits offer a simplified workflow similar to standard IHC staining methods. After detecting the antigen with a primary antibody, a secondary antibody or streptavidin (for biotinylated primary antibodies) conjugated with poly-HRP is used for staining. The HRP enzyme activates a chromogen tyramide, leading to localized covalent deposition of the tyramide derivative at tyrosine residues surrounding the targeted protein epitope. Treatment with the EverRed or EverBlue reagent then generates a permanent colorimetric signal at the site of the covalent tyramide deposition.
Table 4. SuperBoost EverRed and EverBlue reagent selection guide
| SuperBoost EverRed HRP Kit | SuperBoost EverBlue HRP Kit | |
| Detection | Colorimetric and fluorescent | |
| Colorimetric readout | Red color | Blue color |
| Fluorescence readout | TRITC/RFP | Cy5 |
| Sample type | Tissue | |
| Application | Single or multiplex permanent colorimetric staining for IHC | |
| Key benefits |
| |
| HRP Substrate Kits* | E40963 E40971 | E40964 E40972 |
| SuperBoost Kits: goat anti-mouse | E40965 | E40966 |
| SuperBoost Kits: goat anti rabbit | E40967 | E40968 |
| SuperBoost kits: Streptavidin | E40969 | E40970 |
*The HRP substate kits include the tyramide HRP substrate and SuperBoost EverRed or EverBlue reagent for permanent red or blue colorimetric detection in IHC using your own HRP conjugate (not provided).
Table 5. Ordering information for stand-alone poly HRP antibodies
SuperBoost EverRed and EverBlue experimental results
Figure 3. FFPE-preserved human tonsil tissue was processed for immunohistochemistry and labeled with rabbit anti-Ki67 primary antibody, which was detected with the SuperBoost (A) EverRed Goat anti-Rabbit IgG (Cat. No. E40967), (B) EverBlue Goat anti-Rabbit IgG (Cat. No. E40968), or (C) DAB using SuperBoost Goat anti-Rabbit Poly HRP IgG (Cat. No. B40962). Images were taken using an EVOS M7000 Imaging System (Cat. No. AMF7000) with 4X objective.
Figure 4. Cryo-preserved human tonsil tissue was processed for immunohistochemistry and labeled with rabbit anti-Ki67 primary antibody and mouse anti-KRT15 primary antibody, which was detected with DAB (ImmPACT DAB kit, OEM from Vector Laboratories) using HRP-goat anti-rabbit IgG and then with the SuperBoost (A) EverRed Goat anti-Mouse IgG (Cat. No. E40965) or (B) EverBlue Goat anti-Mouse IgG (Cat. No. E40966). Images were taken using an EVOS M7000 Imaging System (Cat. No. AMF7000) with 20X objective.
Figure 5. FFPE-preserved human breast tissue was processed for immunohistochemistry and labeled with anti-ESR1 primary antibody, which was detected with the SuperBoost EverRed Goat anti-Rabbit IgG (Cat. No. E40967) or EverBlue Goat anti-Rabbit IgG (Cat. No. E40968). Images were taken with an EVOS M7000 Imaging System (Cat. No. AMF7000) 40X objective using brightfield, EVOS Light Cube, RFP 2.0 (Cat. No. AMEP4952) or EVOS Light Cube, Cy5 2.0 (Cat. No. AMEP4956) using the same exposure and gain.
What is tyramide signal amplification?
Tyramide signal amplification is a technique used to enhance the detection of low-abundance targets in imaging applications based on the enzymatic deposition of fluorescent or chromogenic reporter molecules onto the target of interest. Alexa Fluor Tyramide SuperBoost kits and reagents are specifically designed for exceptional signal amplification, offering additional signal definition and clarity required for fluorescent imaging of low-abundance targets. SuperBoost EverRed and EverBlue colorimetric kits and reagents can also be used for permanent staining in colorimetric IHC experiments for multiplexing with DAB substrate or as an alternative colorimetric stain.
How does tyramide signal amplification work?
Tyramide signal amplification used in the SuperBoost kits utilizes the catalytic activity of horseradish peroxidase (HRP) for high-density labeling of a target protein or nucleic acid sequence in situ. Typical immunocytochemistry (ICC), immunohistochemistry (IHC) and in situ hybridization (ISH) experiments using the SuperBoost tyramide reagents require 10–5000 times less primary antibody than standard ICC/IHC/ISH experiments to achieve the same signal intensity. Since the SuperBoost kits greatly enhance specific signal intensity over background, they can be easily optimized to detect specific signal in samples where high endogenous autofluorescence is observed.
The tyramide signal amplification process includes the use of horseradish peroxidase (HRP) to enzymatically convert fluorophore or chromogen tyramides to covalently 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.
Click image to enlarge
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).
Benefits of tyramide signal amplification and deposition
- Enable excellent detection of low-abundance targets due to poly HRP secondary antibody
- Ability to label with antibodies from the same species due to covalent attachment of the fluorophore, allowing antibody stripping in between rounds of tyramide signal amplification
- High sensitivity allows the use of significantly lower concentrations of primary antibody
Table 1. Invitrogen tyramide signal amplification and deposition reagent options
| Alexa Fluor Tyramide SuperBoost Reagents | SuperBoost EverRed and EverBlue Tyramide Reagents | |
|---|---|---|
| Detection | Fluorescence | Colorimetric and fluorescence |
| Use | Single or 2-plex staining for fluorescence ICC, IHC, and ISH | Single or multiplex permanent colorimetric staining for IHC |
| Brightness | Higher | High |
| Sample types | Cells or Tissues | Tissues |
Alexa Fluor Tyramide SuperBoost kits for signal amplification for ICC/IHC
Combining the brightness of Invitrogen Alexa Fluor dyes with poly-HRP–mediated tyramide signal amplification, SuperBoost reagents yield precision and sensitivity 10–200 times greater than standard ICC/ IHC/ISH and 2–10 times that of other tyramide amplification techniques.
Benefits of the SuperBoost tyramide signal amplification kits 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 does SuperBoost tyramide signal amplification work?
Excellent for 1 or 2 plex imaging, Alexa Fluor Tyramide SuperBoost 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.
Click image to enlarge
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.
Examples of SuperBoost tyramide based amplification in IF, IHC, and ISH
Alexa Fluor Tyramide SuperBoost kits are compatible with a range of other marker detection and cell staining techniques, enabling multiplex experiments and fluorescence colocalization studies. These kits work with cell types and fluorescence imaging systems commonly used in standard ICC, IHC, and ISH 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
- Additional Alexa Fluor Tyramide SuperBoost kits
Table 2. Alexa Fluor Tyramide SuperBoost reagent selection guide
| Alexa Fluor 350 Tyramide Reagent | Alexa Fluor 488 Tyramide Reagent | Alexa Fluor 546 Tyramide Reagent | Alexa Fluor 555 Tyramide Reagent | Alexa Fluor 568 Tyramide Reagent | Alexa Fluor 594 Tyramide Reagent | Alexa Fluor 647 Tyramide Reagent | Alexa Fluor Plus 750 Tyramide Reagent | Biotin-XX | |
|---|---|---|---|---|---|---|---|---|---|
| Detection | Fluorescence | ||||||||
| Ex/Em | 347/442 | 495/519 | 556/573 | 555/565 | 579/604 | 591/617 | 650/668 | 750/790 | N/A |
| Channel | 430 | 488 | 514 | 555 | 594 | 594 | 647 | 750 | N/A |
| EVOS filter | CFP | GFP | YFP | RFP | Texas Red | Texas Red | Cy5 | Cy7 | N/A |
| Application | Single or 2-plex staining for fluorescence ICC, IHC, and ISH | ||||||||
| Key benefits |
| ||||||||
| Tyramide Reagent | B40952 | B40953 | B40954 | B40955 | B40956 | B40957 | B40958 | B56131 | B40951 |
| SuperBoost Kits: goat anti-mouse | B40912 B40941 (50 coverslips) | B40913 | B40915 B40942 (50 coverslips) | B40916 | B40911 | ||||
| SuperBoost Kits: goat anti-rabbit | B40922 B40943 (50 coverslips) | B40923 | B40925 B40944 (50 coverslips) | B40926 | B40921 | ||||
| SuperBoost kits: Streptavidin | B40932 | B40933 | B40935 | B40921 | B40931 | ||||
Table 3. Ordering information for stand-alone poly HRP antibodies
Alexa Fluor Tyramide SuperBoost experimental results
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 labeled with 3 different primary antibodies: rabbit anti-H2B detected with Alexa Fluor 647 Tyramide SuperBoost Kit (green), rabbit anti–smooth muscle actin antibody detected with Alexa Fluor 488 Tyramide SuperBoost Kit (red), and rabbit anti-Ki67 antibody detected with Alexa Fluor 594 Tyramide SuperBoost Kit (blue).
With ICC in cell culture
Sample type: Fixed and permeabilized HeLa cells.
Antibodies: Immunolabeled sequentially with 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–tubulin and 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 and an anti–β-catenin 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.
SuperBoost EverRed and EverBlue kits for permanent colorimetric IHC
SuperBoost colorimetric signal anchoring, achieved with SuperBoost EverRed and EverBlue Colorimetric HRP Kits, enables highly sensitive and permanent target detection in multiplexable colorimetric IHC experiments. It combines poly-HRP-mediated tyramide signal deposition with SuperBoost EverRed and EverBlue for precise and sensitive colorimetric tissue staining. These SuperBoost kits use HRP catalytic activity for high-density labeling, resulting in wash-resistant, mountable, and archivable staining. SuperBoost EverRed and EverBlue provide sharp and intense signals without the need for air-drying or unconventional protocols. These user-friendly kits work with various tissue types and can be imaged with any brightfield microscope, yielding high-resolution images.
Benefits of the SuperBoost EverRed and EverBlue HRP Kits include:
- Permanent colorimetric staining that is also fluorescent, allowing confirmation of the staining pattern with both brightfield and fluorescence imaging
- Compatibility with other common colorimetric counterstains
- Multiplexable between EverRed, EverBlue, and DAB
How does SuperBoost color signal anchoring work?
SuperBoost EverRed and EverBlue HRP Kits offer a simplified workflow similar to standard IHC staining methods. After detecting the antigen with a primary antibody, a secondary antibody or streptavidin (for biotinylated primary antibodies) conjugated with poly-HRP is used for staining. The HRP enzyme activates a chromogen tyramide, leading to localized covalent deposition of the tyramide derivative at tyrosine residues surrounding the targeted protein epitope. Treatment with the EverRed or EverBlue reagent then generates a permanent colorimetric signal at the site of the covalent tyramide deposition.
Table 4. SuperBoost EverRed and EverBlue reagent selection guide
| SuperBoost EverRed HRP Kit | SuperBoost EverBlue HRP Kit | |
| Detection | Colorimetric and fluorescent | |
| Colorimetric readout | Red color | Blue color |
| Fluorescence readout | TRITC/RFP | Cy5 |
| Sample type | Tissue | |
| Application | Single or multiplex permanent colorimetric staining for IHC | |
| Key benefits |
| |
| HRP Substrate Kits* | E40963 E40971 | E40964 E40972 |
| SuperBoost Kits: goat anti-mouse | E40965 | E40966 |
| SuperBoost Kits: goat anti rabbit | E40967 | E40968 |
| SuperBoost kits: Streptavidin | E40969 | E40970 |
*The HRP substate kits include the tyramide HRP substrate and SuperBoost EverRed or EverBlue reagent for permanent red or blue colorimetric detection in IHC using your own HRP conjugate (not provided).
Table 5. Ordering information for stand-alone poly HRP antibodies
SuperBoost EverRed and EverBlue experimental results
Figure 3. FFPE-preserved human tonsil tissue was processed for immunohistochemistry and labeled with rabbit anti-Ki67 primary antibody, which was detected with the SuperBoost (A) EverRed Goat anti-Rabbit IgG (Cat. No. E40967), (B) EverBlue Goat anti-Rabbit IgG (Cat. No. E40968), or (C) DAB using SuperBoost Goat anti-Rabbit Poly HRP IgG (Cat. No. B40962). Images were taken using an EVOS M7000 Imaging System (Cat. No. AMF7000) with 4X objective.
Figure 4. Cryo-preserved human tonsil tissue was processed for immunohistochemistry and labeled with rabbit anti-Ki67 primary antibody and mouse anti-KRT15 primary antibody, which was detected with DAB (ImmPACT DAB kit, OEM from Vector Laboratories) using HRP-goat anti-rabbit IgG and then with the SuperBoost (A) EverRed Goat anti-Mouse IgG (Cat. No. E40965) or (B) EverBlue Goat anti-Mouse IgG (Cat. No. E40966). Images were taken using an EVOS M7000 Imaging System (Cat. No. AMF7000) with 20X objective.
Figure 5. FFPE-preserved human breast tissue was processed for immunohistochemistry and labeled with anti-ESR1 primary antibody, which was detected with the SuperBoost EverRed Goat anti-Rabbit IgG (Cat. No. E40967) or EverBlue Goat anti-Rabbit IgG (Cat. No. E40968). Images were taken with an EVOS M7000 Imaging System (Cat. No. AMF7000) 40X objective using brightfield, EVOS Light Cube, RFP 2.0 (Cat. No. AMEP4952) or EVOS Light Cube, Cy5 2.0 (Cat. No. AMEP4956) using the same exposure and gain.
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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