Cy7 tyramide

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. The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label translates ultrasensitive detection of low-abundance targets and the use of smaller amounts of antibodies and hybridization probes. In immunohistochemical applications, sensitivity enhancements derived from TSA method allow primary antibody dilutions to be increased to reduce nonspecific background signals, and can overcome weak immunolabeling caused by suboptimal fixation procedures or low levels of target expression. Cy7 tyramide contains the popular Cy7 fluorophore that can be readily detected with the standard Cy7 filter set. Its NIR excitation and emission make the probe an ideal choice for the applications where the common tyramides may have an interference resulted from the inherent fluorescence of tissues or other samples.

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.

Tyramide stock solution (200X)

Add appropriate amount of DMSO to make 1 or 2 mg/mL stock solution and mix well. Note: Unused Tyramide stock solution can be stored at 2-8 °C.

PREPARATION OF WORKING SOLUTION

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 similar performance. Note: Tyramide working solution should be used immediately and made fresh on the day of use. Note: Concentration should be optimized initially for final appropriate concentration for the 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-tissueimmunohistochemistry-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

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
Cy7 Styramide Superior Replacement for Cy7 tyramide	756	779	250000	0.3	0.05	0.036
Cy7 tetrazine	756	779	250000	0.3	0.05	0.036
Cy3 tyramide	555	569	150000¹	0.15¹	0.07	0.073
Cy5 tyramide	651	670	250000¹	0.27¹, 0.4²	0.02	0.03
XFD514 tyramide	518	543	80000	-	0.31	0.18
XFD532 tyramide	534	553	81000	0.61¹	0.24	0.09
Fluorescein Tyramide	498	517	80000¹	0.7900¹, 0.95²	0.32	0.35
PerCP-Cy7	757	783	350000	-	-	-

Citations

View all 1 citations: Citation Explorer

Anti-inflammatory effects of MerTK by inducing M2 macrophage polarization via PI3K/Akt/GSK-3$\beta$ pathway in gout
Authors: Chen, Fangfang and Li, Yixuan and Zhao, Li and Lin, Cong and Zhou, Yingzi and Ye, Wenjing and Wan, Weiguo and Zou, Hejian and Xue, Yu
Journal: International Immunopharmacology (2024): 112942

References

View all 50 references: Citation Explorer

Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures.
Authors: Schafer, Johanna M and Pietenpol, Jennifer A
Journal: Bio-protocol (2020): e3677

Cascade signal amplification for sensitive detection of exosomes by integrating tyramide and surface-initiated enzymatic polymerization.
Authors: Huang, Zhipeng and Lin, Qiuyuan and Yang, Bin and Ye, Xin and Chen, Hui and Weng, Wenhao and Kong, Jilie
Journal: Chemical communications (Cambridge, England) (2020): 12793-12796

Detection of Cytokine Receptors Using Tyramide Signal Amplification for Immunofluorescence.
Authors: Wang, Herui and Pangilinan, Ryan L and Zhu, Yan
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 89-97

Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.
Authors: Mori, Hidetoshi and Bolen, Jennifer and Schuetter, Louis and Massion, Pierre and Hoyt, Clifford C and VandenBerg, Scott and Esserman, Laura and Borowsky, Alexander D and Campbell, Michael J
Journal: Journal of mammary gland biology and neoplasia (2020): 417-432

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies.
Authors: Parra, Edwin Roger and Jiang, Mei and Solis, Luisa and Mino, Barbara and Laberiano, Caddie and Hernandez, Sharia and Gite, Swati and Verma, Anuj and Tetzlaff, Michael and Haymaker, Cara and Tamegnon, Auriole and Rodriguez-Canales, Jaime and Hoyd, Clifford and Bernachez, Chantale and Wistuba, Ignacio
Journal: Cancers (2020)

Page updated on November 19, 2024

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Catalog Number	11064
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Physical properties

Molecular weight	903.21
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.05
Correction Factor (280 nm)	0.036
Correction Factor (482 nm)	0.0005
Correction Factor (565 nm)	0.0193
Correction Factor (650 nm)	0.165
Extinction coefficient (cm ^-1 M ^-1)	250000
Excitation (nm)	756
Emission (nm)	779
Quantum yield	0.3

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