iFluor® 610 Styramide

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. iFluor® 610 Styramide is a new unique red fluorescent PSA reagent for multicolor application with our existing PSA and TSA reagents. AAT Bioquest offers the largest collection of TSA regents. We are the exclusive source of the superior PSA reagents for multicolor applications.

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™ 610 Styramide stock solution (100X)

Add 100 µL of DMSO into the vial of iFluor™ 610 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™ 610 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® 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® 660 Styramide	663	678	250000¹	0.26¹	0.07	0.08
iFluor® 405 Styramide	403	427	37000¹	0.91¹	0.48	0.77
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 (12)

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 12 references: Citation Explorer

Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.
Authors: Jessop, Peter and Gering, Martin
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 193-208

Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation.
Authors: Kuwajima, Masaaki and Ostrovskaya, Olga I and Cao, Guan and Weisberg, Seth A and Harris, Kristen M and Zemelman, Boris V
Journal: PloS one (2020): e0226797

Intensive Immunofluorescence Staining Methods for Low Expression Protein: Detection of Intestinal Stem Cell Marker LGR5.
Authors: Yamazaki, Masaki and Kato, Atsuhiko and Zaitsu, Yoko and Watanabe, Takeshi and Iimori, Makoto and Funahashi, Shinichi and Kitao, Hiroyuki and Saeki, Hiroshi and Oki, Eiji and Suzuki, Masami
Journal: Acta histochemica et cytochemica (2015): 159-64

Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.
Authors: Clutter, Matthew R and Heffner, Garrett C and Krutzik, Peter O and Sachen, Kacey L and Nolan, Garry P
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2010): 1020-31

Methoxychlor and estradiol induce oxidative stress DNA damage in the mouse ovarian surface epithelium.
Authors: Symonds, Daniel A and Merchenthaler, Istvan and Flaws, Jodi A
Journal: Toxicological sciences : an official journal of the Society of Toxicology (2008): 182-7

Page updated on November 16, 2024

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Catalog Number	44904
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Physical properties

Molecular weight	1321.64
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.32
Correction Factor (280 nm)	0.49
Extinction coefficient (cm ^-1 M ^-1)	110000¹
Excitation (nm)	610
Emission (nm)	628
Quantum yield	0.85¹

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