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iFluor® 610 Styramide

Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using iFluor® 610 styramide (Cat No. 44904) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using iFluor® 610 styramide (Cat No. 44904) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
Ordering information
Price ()
Catalog Number44904
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight1321.64
SolventDMSO
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 yield0.851
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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iFluor® 514 succinimidyl ester
iFluor® 532 succinimidyl ester
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iFluor® 594 succinimidyl ester
iFluor® 633 succinimidyl ester
iFluor® 647 succinimidyl ester
iFluor® 660 succinimidyl ester
iFluor® 680 succinimidyl ester
iFluor® 700 succinimidyl ester
iFluor® 750 succinimidyl ester
iFluor® 610 succinimidyl ester
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iFluor® 800 succinimidyl ester
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iFluor® 430 succinimidyl ester
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Show More (246)

OverviewpdfSDSpdfProtocol


Molecular weight
1321.64
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
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.

Platform


Fluorescence microscope

ExcitationCy3/TRITC filter set
EmissionCy3/TRITC filter set
Recommended plateBlack wall/clear bottom

Example protocol


AT A GLANCE

Protocol Summary
  1. Fix/permeabilize/block cells or tissue
  2. Add primary antibody in blocking buffer
  3. Add HRP-conjugated secondary antibody
  4. 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
  1. Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
  2. Rinse the cells or tissue with PBS twice.
  3. Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
  4. 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
  1. 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.
  2. Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.
  3. Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.
  4. Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4 °C.
  5. Wash with PBS three times for 5 minutes each.
  6. 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.
  7. Wash with PBS three times for 5 minutes each. 

Styramide labeling
  1. 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.
  2. Rinse with PBS three times. 

Counterstain and fluorescence imaging
  1. 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.
  2. Mount the coverslip using a mounting medium with anti-fading properties.
  3. 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® 610 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 mg0.5 mg1 mg5 mg10 mg
1 mM75.664 µL378.318 µL756.636 µL3.783 mL7.566 mL
5 mM15.133 µL75.664 µL151.327 µL756.636 µL1.513 mL
10 mM7.566 µL37.832 µL75.664 µL378.318 µL756.636 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

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 yield0.851

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 Styramide *Superior Replacement for Alexa Fluor 350 tyramide*3454502000010.9510.830.23
iFluor® 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide and Opal 520*4915167500010.910.210.11
iFluor® 546 Styramide *Superior Replacement for Alexa Fluor 546 tyramide*54155710000010.6710.250.15
iFluor® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570*55757010000010.6410.230.14
iFluor® 568 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*56858710000010.5710.340.15
iFluor® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide*58860418000010.5310.050.04
iFluor® 647 Styramide *Superior Replacement for Alexa Fluor 647 tyramide*65667025000010.2510.030.03
iFluor® 680 Styramide *Superior Replacement for Alexa Fluor 680 tyramide and Opal 690*68470122000010.2310.0970.094
iFluor® 700 Styramide *Superior Replacement for Alexa Fluor 700 tyramide*69071322000010.2310.090.04
iFluor® 750 Styramide *Superior Replacement for Alexa Fluor 750 tyramide*75777927500010.1210.0440.039
iFluor® 790 Styramide *Superior Replacement for Alexa Fluor 790 tyramide*78781225000010.1310.10.09
iFluor® 450 Styramide *Superior Replacement for Opal Polaris 480*4515024000010.8210.450.27
iFluor® 514 Styramide *Superior Replacement for Opal 540*5115277500010.8310.2650.116
iFluor® 532 Styramide5375609000010.6810.260.16
iFluor® 633 Styramide *Superior Replacement for Opal 650*64065425000010.2910.0620.044
iFluor® 440 Styramide4344804000010.6710.3520.229
iFluor® 460 Styramide468493800001~0.810.980.46
iFluor® 660 Styramide66367825000010.2610.070.08
iFluor® 405 Styramide4034273700010.9110.480.77
Show More (20)

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
Genotyping of phenotypically defined cells in neoplasia: enhanced immunoFISH via tyramide signal amplification (TSA) segregates immunophenotypically-defined cell populations for gated genotyping.
Authors: Tubbs, Raymond R and Das, Kingshuk and Cook, James R and Pettay, James D and Roche, Patrick C and Grogan, Thomas
Journal: Journal of molecular histology (2007): 129-34
A CARD-FISH protocol for the identification and enumeration of epiphytic bacteria on marine algae.
Authors: Tujula, Niina A and Holmström, Carola and Mussmann, Marc and Amann, Rudolf and Kjelleberg, Staffan and Crocetti, Gregory R
Journal: Journal of microbiological methods (2006): 604-7
Novel oxidative self-anchoring fluorescent substrates for the histochemical localization of endogenous and immunobound peroxidase activity.
Authors: Krieg, Reimar and Halbhuber, Karl-Jürgen
Journal: Journal of molecular histology (2004): 471-87
Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.
Authors: Warsen, Adelaide E and Krug, Melissa J and LaFrentz, Stacey and Stanek, Danielle R and Loge, Frank J and Call, Douglas R
Journal: Applied and environmental microbiology (2004): 4216-21
Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.
Authors: Panicker, Gitika and Call, Douglas R and Krug, Melissa J and Bej, Asim K
Journal: Applied and environmental microbiology (2004): 7436-44