iFluor® 450 Tyramide *Superior Replacement for Opal 480*
For many immunohistochemical (IHC) applications, traditional enzymatic amplification procedures are sufficient for achieving adequate antigen detection. However, several factors limit their sensitivity and utility. Tyramide signal amplification (TSA) has proven to be a particularly versatile and powerful enzyme amplification technique with improved assay sensitivity. TSA is based on the ability of HRP, in the presence of low concentrations of hydrogen peroxide, to convert labeled tyramine-containing substrate into an oxidized, highly reactive free radical that can covalently bind to tyrosine residues at or near the HRP. To achieve maximal IHC detection, tyramine is prelabeled with a fluorophore. The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label results in the ability to detect low-abundance targets with ultrasensitive precision and reduces the amount of antibodies and hybridization probes needed. In IHC applications, this method can also enhance sensitivity in cases where the primary antibody dilution needs to be increased to reduce nonspecific background signals or overcome weak immunolabeling due to suboptimal fixation procedures or low levels of target expression. The iFluor® 450 tyramide contains the bright iFluor® 450 that can be readily detected with the standard FITC filter set. The iFluor® dyes offer brighter fluorescence intensities, increased photostability, and enhanced water solubility, leading to fluorescence signals with significantly higher precision and sensitivity. iFluor® 450 is an excellent replacement for Opal 480 or other spectrally similar fluorescent tyramide conjugates.
Example protocol
AT A GLANCE
Protocol Summary
- Fix/permeabilize/block cells or tissue
- Add primary antibody in blocking buffer
- Add HRP-conjugated secondary antibody
- Prepare tyramide working solution and apply in cells or tissue for 5-10 minutes at room temperature
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note Unused Tyramide stock solution can be stored at 2-8 °C.
iFluor™ 450 Tyramide stock solution (200X)
Add 100 µL DMSO to vial and mix well.Note Unused Tyramide stock solution can be stored at 2-8 °C.
PREPARATION OF WORKING SOLUTION
iFluor™ 450 Tyramide working solution (1X)
Add 100 µL of Tyramide stock solution into 20 mL of buffer of your choice containing 0.003% H2O2.Note Tris Buffer, pH=7.4 can be used for optimal performance.
Note Tyramide working solution should be used immediately and made fresh on the day of use.
Note 20 mL solution is good for 200 tests.
SAMPLE EXPERIMENTAL PROTOCOL
This protocol is applicable for both cells and tissues staining.
Protocol can be found at
https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html
Cell fixation and permeabilization
- Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
- Rinse the cells or tissue with PBS twice.
- Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
- Rinse the cells or tissue with PBS twice.
Tissue fixation, deparaffinization and rehydration
Deparaffinize and dehydrate the tissue according to the standard IHC protocols. Perform antigen retrieval with preferred specific solution/protocol as needed.Protocol can be found at
https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html
Peroxidase labeling
- Optional: Quench endogenous peroxidase activity by incubating cell or tissue sample in peroxidase quenching solution (such as 3% hydrogen peroxide) for 10 minutes. Rinse with PBS twice at room temperature.
- Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.
- Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.
- Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4 °C.
- Wash with PBS three times for 5 minutes each.
- Apply 100 µL of secondary antibody-HRP working solution to each sample and incubate for 60 minutes at room temperature.
Note Incubation time and concentration can be varied depending on the signal intensity. - Wash with PBS three times for 5 minutes each.
Tyramide labeling
- Prepare and apply 100 µL of tyramide working solution to each sample and incubate for 5-10 minutes at room temperature.
Note If you observe non-specific signal, you can shorten the incubation time with tyramide. You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use lower concentration of tyramide in the working solution. - Rinse with PBS three times.
Counterstain and fluorescence imaging
- Counterstain the cell or tissue samples as needed. AAT provides a series of nucleus counterstain reagents as listed in Table 1. Follow the instruction provided with the reagents.
- Mount the coverslip using a mounting medium with anti-fading properties.
- Use the appropriate filter set to visualize the signal from the tyramide labeling.
Cat# | Product Name | Ex/Em (nm) |
17548 | Nuclear Blue™ DCS1 | 350/461 |
17550 | Nuclear Green™ DCS1 | 503/526 |
17551 | Nuclear Orange™ DCS1 | 528/576 |
17552 | Nuclear Red™ DCS1 | 642/660 |
Spectrum
Open in Advanced Spectrum Viewer
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
iFluor® 488 tyramide | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
iFluor® 450 maleimide | 451 | 502 | 400001 | 0.821 | 0.45 | 0.27 |
iFluor® 555 Tyramide | 557 | 570 | 1000001 | 0.641 | 0.23 | 0.14 |
iFluor® 647 Tyramide | 656 | 670 | 2500001 | 0.251 | 0.03 | 0.03 |
iFluor® 350 Tyramide | 345 | 450 | 200001 | 0.951 | 0.83 | 0.23 |
iFluor® 546 Tyramide | 541 | 557 | 1000001 | 0.671 | 0.25 | 0.15 |
iFluor® 568 Tyramide | 568 | 587 | 1000001 | 0.571 | 0.34 | 0.15 |
iFluor® 594 Tyramide | 587 | 603 | 2000001 | 0.531 | 0.05 | 0.04 |
iFluor® 450 Styramide *Superior Replacement for Opal Polaris 480* | 451 | 502 | 400001 | 0.821 | 0.45 | 0.27 |
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References
View all 1 references: Citation Explorer
Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study.
Authors: Taube, Janis M and Roman, Kristin and Engle, Elizabeth L and Wang, Chichung and Ballesteros-Merino, Carmen and Jensen, Shawn M and McGuire, John and Jiang, Mei and Coltharp, Carla and Remeniuk, Bethany and Wistuba, Ignacio and Locke, Darren and Parra, Edwin R and Fox, Bernard A and Rimm, David L and Hoyt, Cliff
Journal: Journal for immunotherapy of cancer (2021)
Authors: Taube, Janis M and Roman, Kristin and Engle, Elizabeth L and Wang, Chichung and Ballesteros-Merino, Carmen and Jensen, Shawn M and McGuire, John and Jiang, Mei and Coltharp, Carla and Remeniuk, Bethany and Wistuba, Ignacio and Locke, Darren and Parra, Edwin R and Fox, Bernard A and Rimm, David L and Hoyt, Cliff
Journal: Journal for immunotherapy of cancer (2021)
Page updated on October 4, 2024