Cy7 Styramide Superior Replacement for Cy7 tyramide

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Additional ordering information

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

Physical properties

Molecular weight	1062.40
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

Direct upgrades

iFluor® 750 Styramide *Superior Replacement for Alexa Fluor 750 tyramide*

Overview SDS Protocol

Upgrade: iFluor® 750 Styramide *Superior Replacement for Alexa Fluor 750 tyramide*

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 Styramide™ 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.

1. Cy7 Styramide stock solution (100X)

Add 100 µL of DMSO into the vial of Cy7 Styramide conjugate to make 100X Styramide stock solution.
Note Make single use aliquots, and store unused 100X stock solution at 2-8 °C in dark place and avoid repeat freeze-thaw cycles.

2. H₂O₂ stock solution

Add 10 µL of 3% hydrogen peroxide (Not provided) to 90 µL of ddH₂O.
Note Prepare the 100X H₂O₂ solution fresh on the day of use.

PREPARATION OF WORKING SOLUTION

1. Cy7 Styramide working solution (1X)

Every 1 mL of Reaction Buffer requires 10 µL of Styramide™ stock solution and 10 µL of H₂O₂ stock solution.
Note The Styramide provided is enough for 100 tests based on 100 µL of Styramide working solution needed per coverslip or per well in a 96-well microplate.
Note The Styramide working solution must be used within 2 hours after preparation and avoid direct exposure to light.

2. Secondary antibody-HRP working solution

Make appropriate concentration of secondary antibody-HRP working solution as per the manufacturer's recommendations.

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.

Styramide labeling

Prepare and apply 100 µL of Styramide 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 Styramide. You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use lower concentration of Styramide 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 Styramide 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 Styramide *Superior Replacement for 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	94.127 µL	470.633 µL	941.265 µL	4.706 mL	9.413 mL
5 mM	18.825 µL	94.127 µL	188.253 µL	941.265 µL	1.883 mL
10 mM	9.413 µL	47.063 µL	94.127 µL	470.633 µL	941.265 µL

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)
Cy7 tyramide	756	779	250000
Cy7 tetrazine	756	779	250000
PerCP-Cy7	757	783	350000

Images

Figure 1. Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA ™ amplified methods. Human lung adenocarcinoma positive tissue sections were stained with Mouse anti-EpCAM or Control Mouse IgG antibody and then incubated with polyHRP-labeled Goat anti-Mouse IgG secondary antibody followed by Cy7 Styramide™ (Cat#45066).

References

View all 50 references: Citation Explorer

Somatostatin receptor profile in pituitary thyrotroph adenomas.
Authors: Thodou, Eleni and Kontogeorgos, George
Journal: Clinical neurology and neurosurgery (2020): 105865

Identification of Murine Basophils by Flow Cytometry and Histology.
Authors: Schwartz, Christian and Voehringer, David
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 367-375

Use of TAI-FISH to visualize neural ensembles activated by multiple stimuli.
Authors: Zhang, Qi and He, Qiye and Wang, Jihua and Fu, Chaoying and Hu, Hailan
Journal: Nature protocols (2018): 118-133

Selective Inhibition of Amygdala Neuronal Ensembles Encoding Nicotine-Associated Memories Inhibits Nicotine Preference and Relapse.
Authors: Xue, Yan-Xue and Chen, Ya-Yun and Zhang, Li-Bo and Zhang, Li-Qun and Huang, Geng-Di and Sun, Shi-Chao and Deng, Jia-Hui and Luo, Yi-Xiao and Bao, Yan-Ping and Wu, Ping and Han, Ying and Hope, Bruce T and Shaham, Yavin and Shi, Jie and Lu, Lin
Journal: Biological psychiatry (2017): 781-793

Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy.
Authors: Hisada, Yohei and Auriemma, Alyson C and Alexander, Wyeth and Ay, Cihan and Mackman, Nigel
Journal: Thrombosis research (2017): 65-72

Immunohistochemical Methods for Measuring Tissue Lymphangiogenesis.
Authors: Royston, Daniel J and Clasper, Steven and Jackson, David G
Journal: Methods in molecular biology (Clifton, N.J.) (2016): 35-48

Development of a highly sensitive immunohistochemical method to detect neurochemical molecules in formalin-fixed and paraffin-embedded tissues from autopsied human brains.
Authors: Goto, Satoshi and Morigaki, Ryoma and Okita, Shinya and Nagahiro, Shinji and Kaji, Ryuji
Journal: Frontiers in neuroanatomy (2015): 22

TRPM8 has a key role in experimental colitis-induced visceral hyperalgesia in mice.
Authors: Hosoya, T and Matsumoto, K and Tashima, K and Nakamura, H and Fujino, H and Murayama, T and Horie, S
Journal: Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society (2014): 1112-21

Visualizing an emotional valence map in the limbic forebrain by TAI-FISH.
Authors: Xiu, Jianbo and Zhang, Qi and Zhou, Tao and Zhou, Ting-ting and Chen, Yang and Hu, Hailan
Journal: Nature neuroscience (2014): 1552-9

Expression of E6, p53 and p21 proteins and physical state of HPV16 in cervical cytologies with and without low grade lesions.
Authors: Tagle, Diana K Jiménez and Sotelo, Daniel Hernández and Illades-Aguiar, Berenice and Leyva-Vazquez, Marco A and Alfaro, Eugenia Flores and Coronel, Yaneth Castro and Hernández, Oscar Del Moral and Romero, Luz Del Carmen Alarcón
Journal: International journal of clinical and experimental medicine (2014): 186-93