iFluor® 660 Styramide

Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA<strong> ™</strong> 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 iFluor® 660 Styramide™ (Cat#44906).

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Physical properties

Molecular weight	1462.94
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.08
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	663
Emission (nm)	678
Quantum yield	0.26¹

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

Molecular weight

1462.94

Correction Factor (260 nm)

0.07

Correction Factor (280 nm)

0.08

Extinction coefficient (cm ^-1 M ^-1)

250000¹

Excitation (nm)

663

Emission (nm)

678

Quantum yield

0.26¹

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. 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. iFluor® 660 Styramide is a new unique far red fluorescent PSA reagent for multicolor application with our existing PSA and TSA reagents. Its maximum fluorescence falls between Cy5/Alexa Fluor® 647 and Cy5.5/ Alexa Fluor® 647. AAT Bioquest offers the largest collection of TSA regents. We are the exclusive source of the superior PSA reagents for multicolor applications.

Platform

Fluorescence microscope

Excitation	Cy5 filter set
Emission	Cy5 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. iFluor™ 660 Styramide stock solution (100X)

Add 100 µL of DMSO into the vial of iFluor™ 660 Styramide conjugate to make 100X Styramide stock solution.
Note Make single use aliquots, and store unused 100X stock solution at 2-8 ^oC 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™ 660 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 iFluor® 660 Styramide 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	68.356 µL	341.778 µL	683.555 µL	3.418 mL	6.836 mL
5 mM	13.671 µL	68.356 µL	136.711 µL	683.555 µL	1.367 mL
10 mM	6.836 µL	34.178 µL	68.356 µL	341.778 µL	683.555 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

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Spectral properties

Correction Factor (260 nm)	0.07
Correction Factor (280 nm)	0.08
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	663
Emission (nm)	678
Quantum yield	0.26¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 660 amine	663	678	250000¹	0.26¹	0.07	0.08
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	588	604	180000¹	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® 514 Styramide Superior Replacement for Opal 540	511	527	75000¹	0.83¹	0.265	0.116
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® 405 Styramide	403	427	37000¹	0.91¹	0.48	0.77
iFluor® 660 maleimide	663	678	250000¹	0.26¹	0.07	0.08
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
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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 iFluor® 660 Styramide™ (Cat#44906).

References

View all 4 references: Citation Explorer

Quantum dot-based FRET for sensitive determination of hydrogen peroxide and glucose using tyramide reaction.
Authors: Huang, Xiangyi and Wang, Jinjie and Liu, Heng and Lan, Tao and Ren, Jicun
Journal: Talanta (2013): 79-84

Dual enhancement of triple immunofluorescence using two antibodies from the same species.
Authors: Nakamura, Ayako and Uchihara, Toshiki
Journal: Journal of neuroscience methods (2004): 67-70

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry.
Authors: Pougnard, Claire and Catala, Philippe and Drocourt, Jean-Louis and Legastelois, Stephane and Pernin, Pierre and Pringuez, Emmanuelle and Lebaron, Philippe
Journal: Applied and environmental microbiology (2002): 3102-7

Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors.
Authors: Webb, D R and Bonfiglioli, R G and Carraro, L and Osler, R and Symons, R H
Journal: Phytopathology (1999): 894-901