Cy7 tyramide

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

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

Overview SDS Protocol

Platform

Fluorescence microscope

Excitation	Cy7 filter set
Emission	Cy7 filter set
Recommended plate	Black wall/clear bottom

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

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cy7 tyramide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	110.716 µL	553.581 µL	1.107 mL	5.536 mL	11.072 mL
5 mM	22.143 µL	110.716 µL	221.432 µL	1.107 mL	2.214 mL
10 mM	11.072 µL	55.358 µL	110.716 µL	553.581 µL	1.107 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

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

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	-	-	-

Images

Figure 1. Chemical structure for Cy7 tyramide.

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)

Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced Tyramide Signal Amplification and Its Combination with Immunofluorescent Protein Visualization in Zebrafish.
Authors: Lauter, Gilbert and Söll, Iris and Hauptmann, Giselbert
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 397-409

Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins.
Authors: Dopie, Joseph and Sweredoski, Michael J and Moradian, Annie and Belmont, Andrew S
Journal: The Journal of cell biology (2020)

Highly Sensitive Detection of PCV2 Based on Tyramide Signals and GNPL Amplification.
Authors: Zhang, Shouping and Hu, Bin and Xia, Xiaojing and Xu, Yanzhao and Hang, Bolin and Jiang, Jinqing and Hu, Jianhe
Journal: Molecules (Basel, Switzerland) (2019)

An ultrasensitive electrochemical immunosensor for procalcitonin detection based on the gold nanoparticles-enhanced tyramide signal amplification strategy.
Authors: Liu, Pei and Li, Chao and Zhang, Ruixuan and Tang, Qing and Wei, Jia and Lu, Yan and Shen, Pingping
Journal: Biosensors & bioelectronics (2019): 543-550

A amperometric immunosensor for sensitive detection of circulating tumor cells using a tyramide signal amplification-based signal enhancement system.
Authors: Zhou, Xiaoyan and Li, Yujian and Wu, Haiping and Huang, Wei and Ju, Huangxian and Ding, Shijia
Journal: Biosensors & bioelectronics (2019): 88-94