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.

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.

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.

Table 1.Products recommended for nucleus counterstain.

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
iFluor® 633 tyramide	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 430 Tyramide Superior Replacement for Opal 480	433	498	40000¹	0.78¹	0.68	0.3
iFluor® 680 Tyramide Superior Replacement for Opal 690	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 450 acid	451	502	40000¹	0.82¹	0.45	0.27
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Citations

View all 1 citations: Citation Explorer

Membrane progesterone receptor $\gamma$ (paqr5b) is essential for the formation of neurons in the zebrafish olfactory rosette
Authors: Mustary, Umme Habiba and Maeno, Akiteru and Rahaman, Md Mostafizur and Ali, Md Hasan and Tokumoto, Toshinobu
Journal: Scientific Reports (2024): 24354

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)

Page updated on November 21, 2024

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Catalog Number	45097
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Physical properties

Molecular weight	625.78
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.45
Correction Factor (280 nm)	0.27
Extinction coefficient (cm ^-1 M ^-1)	40000¹
Excitation (nm)	451
Emission (nm)	502
Quantum yield	0.82¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501