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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.
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.
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:
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.
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.
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.
View the immunofluorescence guide to choose appropriate detection technologies.
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:
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).
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.
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).
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.
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.
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. |
Use SlowFade antifade mountant when stripping and reprobing with TSA reagents.
Protocol for SuperBoost tyramide signal and applications including stripping and restaining
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