Fixed and permeabilized HeLa cells, treated using the reagents in the 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.
Superior imaging of low-abundance targets
Offering sensitivity 10–200 times that of standard ICC/IHC/ISH methods, Invitrogen SuperBoost kits are designed for superior signal amplification, definition, and clarity needed for high-resolution imaging of low-abundance targets. Combining the brightness of Invitrogen Alexa Fluor dyes with trusted poly-HRP–mediated tyramide signal amplification, the SuperBoost reagent generates sensitivity typically 2 to 10 times above that of standard treatments, including TSA reagents (PerkinElmer). For imaging that demands definition and clarity, bring your targets out of the background with SuperBoost signal amplification kits.
SuperBoost technology
- The most sensitive fluorescent imaging detection method for low-abundance, hard-to-detect targets
- Easy-to-use kits that produce signals compatible with standard filters
- High-resolution images and multiplex compatibility with fluorescent proteins, DAPI, secondary antibodies, and other SuperBoost kits
Figure 1. Superior sensitivity from SuperBoost kits. 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. 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
Multiplex capability
SuperBoost kits are compatible with a range of other marker detection and cell staining techniques, enabling multiplex experiments and fluorescence colocalization studies. For example, SuperBoost reagent multiplexing can be achieved with:
- Fluorescent markers for counterstaining, such as DAPI
- Fluorescent proteins (i.e., GFP & RFP)
- Standard ICC/IHC
- Other SuperBoost kits
A detailed protocol is available in the product manual.
With GFP and RFP
HeLa cells were treated with Invitrogen CellLight Peroxisome-GFP, BacMam 2.0 to express GFP in peroxisomes. Following treatment with the Invitrogen Image-iT Fixation/Permeabilization Kit, cells were incubated with an anti-prohibitin 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 Invitrogen NucBlue Fixed Cell ReadyProbes Reagent. Images were acquired on a confocal microscope.
With standard ICC/IHC
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
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.
Easy workflow
Compatible with ICC, IHC, and FISH procedures
SuperBoost 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 kits using formaldehyde-fixed cell lines in 2D and 3D cultures, FFPE tissues, and cryosectioned tissues.
Immunocytochemistry (ICC)
Immunohistochemistry (IHC)
Formalin-fixed, paraffin-embedded (FFPE) rat intestinal tissue was incubated with an antibody against histone 2B.This antibody was then detected using the reagents in the Alexa Fluor 594 Tyramide SuperBoost Kit (goat anti–rabbit IgG and Alexa Fluor 594 tyramide). Nuclei were labeled with NucBlue Fixed Cell ReadyProbes Reagent, and samples were mounted in ProLong Diamond Antifade Reagent. Images were acquired and analyzed on an EVOS FL Auto Imaging System.
Fluorescence in situ hybridization (FISH)
U2OS cells were fixed and permeabilized using a standard protocol. To conduct fluorescence in situ hybridization (FISH), cells were then incubated with an hprt gene probe that was detected 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 and analyzed on an EVOS FL Auto Imaging System.
Workflow similar to that of standard ICC/IHC/FISH
SuperBoost kits are based on tryramide signal amplification, which employs the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ. SuperBoost kits offer a simple workflow similar to those used in standard ICC, IHC, and FISH. In this workflow, the fluorophore-conjugated secondary antibodies are replaced with 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 to maintain the specificity and resolution of fluorescent signal.
Figure 2. Workflow for SuperBoost kits. With workflows similar to those of standard ICC/IHC/FISH, these kits can achieve sensitive detection of your targets in a few steps.
When to use tyramide-based amplification
We offer a complete range of detection technologies for fluorescence imaging. Using this suite of tools you can select the optimal technique for your target abundance with the right detection wavelength to multiplex your experiment.
|
| 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. | |||
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