Cy7 Styramide Superior Replacement for Cy7 tyramide

Power Styramide™ Signal Amplification (PSA™) system is one of the most sensitive methods that can detect extremely low-abundance targets in cells and tissues with improved fluorescence signal 10-50 times higher than the widely used tyramide (TSA) reagents. In combination with our superior iFluor® dyes that have higher florescence intensity, increased photostability and enhanced water solubility, the iFluor® dye-labeled Styramide™ conjugates can generate fluorescence signal with significantly higher precision and sensitivity (more than 100 times) than standard ICC/IF/IHC. PSA utilizes the catalytic activity of horseradish peroxidase (HRP) for covalent deposition of fluorophores in situ. PSA radicals have much higher reactivity than tyramide radicals, making the PSA system much faster, more robust and sensitive than the traditional TSA reagents. Compared to tyramide reagents, the Styramide™ conjugates have ability to label the target at higher efficiency and thus generate significantly higher fluorescence signal. Styramide™ conjugates also allow significantly less consumption of primary antibody compared to standard directly conjugate method or tyramide amplification with the same level of sensitivity. Cy7 Styramide is optimized to a superior replacement for Cy7 tyramide (Cy7 TSA) or Alexa Fluor® 750 tyramide or other spectrally similar fluorescent tyramide conjugates or TSA reagents.

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

Spectrum

Open in Advanced Spectrum Viewer

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

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

Page updated on November 21, 2024

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Catalog Number	45066
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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