Learn More
Invitrogen™ Alexa Fluor™ 594 Tyramide SuperBoost™ Kit, streptavidin
Description
SuperBoost kits combine the brightness of AlexaFluor™ dyes with the superior signal amplification of a poly-HRP-mediated tyramide labeling reaction to produce a sensitivity 10-200 times greater than standard methods. For standout research, SuperBoost kits sharpen your results for clear visibility into critical areas that standard imaging methods fail to reveal.
SuperBoost kits are simple to use and easily adapted to standard ICC, IHC, or FISH experimental protocols, using any cell or tissue type. Cells labeled using a SuperBoost kit can be imaged using any type of microscope, producing high-resolution multiplex images. This particular kit features AlexaFluor 594 tyramide (591/617 ex/em), detected using a standard Red/Texas Red™ filter cube. This kit also features HRP-conjugated streptavidin.
• Superior sensitivity for detection of low-level or hard-to-detect targets by fluorescent imaging
• Simple protocol and detection using standard filters
• Suitable for high-resolution multiplex images—co-label with DAPI, secondary antibodies, and other SuperBoost kits
• Requires 10-100 times less primary antibody then standard ICC/IHC/ISH experiments
SuperBoost kits are based on the tyramide signal amplification system, which uses the catalytic activity of horseradish peroxidase (HRP) to generate high density labeling of a target protein or nucleic acid sequence in situ. A typical ICC/IHC/ISH experiment using a SuperBoost kit requires 10-100 times less primary antibody then standard ICC/IHC/ISH experiments. SuperBoost kits offer superior specific signal intensity over background, so the protocol is easily optimized to detect specific signal in samples where high endogenous autofluorescence is observed.
Enhancement of signal using Alexa Fluor tyramides: SuperBoost kits utilize Alexa Fluor tyramides, which react with HRP to ultimately deposit bright and photostable Alexa Fluor dye on surrounding proteins and other similar molecules. SuperBoost kits are the only kits that combine the brightness of Alexa Fluor dyes with the enhancement of tyramide signal amplification to produce a superior signal.
Reaction stop solution: Like any enzyme-based labeling system, it is possible to overdevelop the signal. SuperBoost kits include an HRP stop solution to halt the HRP reaction. HRP stop solution can be used to obtain maximum signal, without increase of background signal. Images produced with optimized HRP reaction times are as sharp as images produced with standard ICC/IHC/ISH methods, but with 10-200 times more sensitivity.
Reduction of background: SuperBoost kits include blockers for the elimination or reduction of endogenous peroxidase and fluorescent background signals. These blockers help ensure that only specific signals are enhanced while keeping non-specific/background signals in check.
Specifications
Specifications
| Conjugate | Alexa Fluor 594 |
| Quantity | 150 Slides |
| Content And Storage | 1 kit sufficient for 150 microscope slides (18 x 18 mm), containing: Blocking buffer (1 X), 22.5 mL |
| Shipping Condition | Approved for shipment at Room Temperature or on Wet Ice |
| Product Line | SuperBoost |
| Product Type | Tyramide Kit |
Frequently Asked Questions (FAQs)
To limit background, we recommend performing a pre-blocking step with 3% H2O2 for 60 mins to inactivate endogenous peroxidases. To limit the localization of labeling, we recommend optimizing the final concentration of the primary and secondary antibodies and the dye-tyramide. You may also limit the incubation time of the dye-tyramide on the sample.
Yes. This involves the sequential application of the antibodies and the tyramides with a HRP-quenching step between antibodies using H2O2.
The SuperBoost tyramide kits utilize poly-HRP labeled antibodies. This provides a greater number of horseradish peroxidase (HRP) molecules per antibody. The original kits used antibodies and streptavidin that were directly conjugated with HRP and thus, limited the number per antibody or streptavidin.
Here are our recommendations:
Use one of our extensive selection of secondary antibodies conjugated to bright, photostable Alexa Fluor dyes. The degree of labeling for each conjugate is 2-8 fluorophores per IgG molecule, with potentially three secondary antibody-binding sites per primary antibody, providing signal amplification of approximately 10-20 fluorophores per primary antibody.
Alternatively, primary antibody labeling can be detected with a biotinylated secondary antibody in conjunction with either a fluorescent streptavidin or a streptavidin bridge followed by a biotinylated reporter such as Qdot biotin. Although processing times increase with additional incubation and endogenous biotin-blocking steps, detection sensitivity also improves as a result of the labeled streptavidin.
For low-abundance targets, signal amplification may be necessary for optimal signal-to-noise ratios. Tyramide signal amplification (TSA) is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate reactive fluorophore-labeled tyramide radicals. These short-lived tyramide radicals covalently couple to nearby residues, producing an amplified fluorescent signal localized at the HRP-target interaction site.
For improved detection sensitivity with rapidly bleaching dyes, our SlowFade Diamond or ProLong Diamond antifade reagents have been shown to increase photostability and reduce initial fluorescence quenching in fixed cells, fixed tissues, and cell-free preparations.
Please review this web page for further optimization tips (https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/bioprobes-journal-of-cell-biology-applications/bioprobes-issues-2011/bioprobes-66-october-2011/guide-to-immunocytochemistry.html).
A common method for amplifying antibody detection is biotin-streptavidin detection, where a biotinylated secondary antibody is combined with subsequent labeling with a dye-conjugated streptavidin. This will amplify the signal by approximately 2-8 times, but endogenous biotin must be blocked beforehand. Another option is to use tyramide-signal amplification, where a horseradish peroxidase conjugate is used with a dye-labeled tyramide. This will amplify the signal by approximately 10-20 times, but endogenous peroxidase will need to be blocked. A final option may be to use a Qdot nanoparticle antibody or streptavidin conjugate, which can yield a signal as much as 40 times higher than a standard organic dye conjugate, depending on the Qdot color.
For Research Use Only. Not for use in diagnostic procedures.