iFluor® 514 Styramide Superior Replacement for Opal 540

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 fluorescence 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 the covalent deposition of fluorophores in situ. PSA radicals have much higher reactivity than tyramide radicals, making the PSA system much faster, more robust, and more sensitive than traditional TSA reagents. Compared to tyramide reagents, the Styramide™ conjugates can label the target at higher efficiency and thus generate a significantly higher fluorescence signal. Styramide™ conjugates also allow substantially less consumption of primary antibodies compared to the standard directly conjugate method or tyramide amplification with the same level of sensitivity. iFluor® 514 Styramide is optimized to be a superior replacement for Opal Polaris 540 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. iFluor™ 514 Styramide stock solution (100X)

Add 100 µL of DMSO into the vial of iFluor™ 514 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.

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. iFluor™ 514 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

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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)
iFluor® 350 Styramide Superior Replacement for Alexa Fluor 350 tyramide	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 488 Styramide Superior Replacement for Alexa Fluor 488 tyramide and Opal 520	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 546 Styramide Superior Replacement for Alexa Fluor 546 tyramide	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 555 Styramide Superior Replacement for Alexa Fluor 555 tyramide and Opal 570	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 568 Styramide Superior Replacement for Alexa Fluor 568 tyramide	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 594 Styramide Superior Replacement for Alexa Fluor 594 tyramide	587	603	200000¹	0.53¹	0.05	0.04
iFluor® 647 Styramide Superior Replacement for Alexa Fluor 647 tyramide	656	670	250000¹	0.25¹	0.03	0.03
iFluor® 680 Styramide Superior Replacement for Alexa Fluor 680 tyramide and Opal 690	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 700 Styramide Superior Replacement for Alexa Fluor 700 tyramide	690	713	220000¹	0.23¹	0.09	0.04
iFluor® 750 Styramide Superior Replacement for Alexa Fluor 750 tyramide	757	779	275000¹	0.12¹	0.044	0.039
iFluor® 790 Styramide Superior Replacement for Alexa Fluor 790 tyramide	787	812	250000¹	0.13¹	0.1	0.09
iFluor® 450 Styramide Superior Replacement for Opal Polaris 480	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 532 Styramide	537	560	90000¹	0.68¹	0.26	0.16
iFluor® 633 Styramide Superior Replacement for Opal 650	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 440 Styramide	434	480	40000¹	0.67¹	0.352	0.229
iFluor® 460 Styramide	468	493	80000¹	~0.8¹	0.98	0.46
iFluor® 610 Styramide	610	628	110000¹	0.85¹	0.32	0.49
iFluor® 660 Styramide	663	678	250000¹	0.26¹	0.07	0.08
iFluor® 405 Styramide	403	427	37000¹	0.91¹	0.48	0.77
iFluor® 514 maleimide	511	527	75000¹	0.83¹	0.265	0.116
iFluor® 570 Styramide Superior Replacement for Alexa Fluor 568 tyramide	557	570	120000¹	0.58¹	-	-
iFluor® 670 Styramide Replacement for Opal 690	671	682	200000¹	0.55¹	0.03	0.033
Show More (13)

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 50 references: Citation Explorer

A novel chemiluminescence imaging immunosensor for prostate specific antigen detection based on a multiple signal amplification strategy.
Authors: Zhao, Li-Zhen and Fu, Yi-Zhuo and Ren, Shu-Wei and Cao, Jun-Tao and Liu, Yan-Ming
Journal: Biosensors & bioelectronics (2021): 112729

Imaging sensor array coupled with dual-signal amplification strategy for ultrasensitive chemiluminescence immunoassay of multiple mycotoxins.
Authors: Zong, Chen and Jiang, Fan and Wang, Xiaoyu and Li, Ping and Xu, Linru and Yang, Hua
Journal: Biosensors & bioelectronics (2021): 112998

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

Page updated on November 16, 2024

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Catalog Number	45022
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Physical properties

Molecular weight	1195.24
Solvent	DMSO

Spectral properties

Absorbance (nm)	511
Correction Factor (260 nm)	0.265
Correction Factor (280 nm)	0.116
Extinction coefficient (cm ^-1 M ^-1)	75000¹
Excitation (nm)	511
Emission (nm)	527
Quantum yield	0.83¹

Storage, safety and handling

Certificate of Origin	Download PDF
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