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iFluor® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide*

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® 594 styramide (Cat No. 45035) 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® 594 styramide (Cat No. 45035) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
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.
Ordering information
Price ()
Catalog Number45035
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 weight1341.71
SolventDMSO
Spectral properties
Absorbance (nm)587
Correction Factor (260 nm)0.05
Correction Factor (280 nm)0.04
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)588
Emission (nm)604
Quantum yield0.531
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
UNSPSC12352200
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iFluor® 700 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 750 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 790 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 350 goat anti-rabbit IgG (H+L)
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iFluor® 488 goat anti-rabbit IgG (H+L)
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iFluor® 594 goat anti-rabbit IgG (H+L)
iFluor® 633 goat anti-rabbit IgG (H+L)
iFluor® 647 goat anti-rabbit IgG (H+L)
iFluor® 680 goat anti-rabbit IgG (H+L)
iFluor® 700 goat anti-rabbit IgG (H+L)
iFluor® 750 goat anti-rabbit IgG (H+L)
iFluor® 790 goat anti-rabbit IgG (H+L)
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iFluor® 405 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 488 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 514 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 810 acid
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iFluor® 820 acid
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iFluor 555™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
iFluor 594™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
iFluor 647™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
iFluor® 840 acid
iFluor® 790-streptavidin conjugate
iFluor® 800-streptavidin conjugate
iFluor® 820-streptavidin conjugate
iFluor® 840-streptavidin conjugate
iFluor® 860-streptavidin conjugate
iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate
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iFluor® 488-Concanavalin A Conjugate
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iFluor® 820 goat anti-mouse IgG (H+L)
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iFluor® 840 goat anti-rabbit IgG (H+L)
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iFluor® 860 goat anti-rabbit IgG (H+L)
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iFluor® 405 succinimidyl ester
iFluor® 488 succinimidyl ester
iFluor® 514 succinimidyl ester
iFluor® 532 succinimidyl ester
iFluor® 555 succinimidyl ester
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
iFluor® 710 succinimidyl ester
iFluor® 790 succinimidyl ester
iFluor® 800 succinimidyl ester
iFluor® 810 succinimidyl ester
iFluor® 820 succinimidyl ester
iFluor® 860 succinimidyl ester
iFluor® 546 succinimidyl ester
iFluor® 568 succinimidyl ester
iFluor® 430 succinimidyl ester
iFluor® 450 succinimidyl ester
iFluor® 840 succinimidyl ester
iFluor® 560 succinimidyl ester
iFluor® 670 succinimidyl ester
iFluor® 460 succinimidyl ester
iFluor® 440 succinimidyl ester
iFluor® 665 succinimidyl ester
iFluor® 690 succinimidyl ester
iFluor® Ultra 594 succinimidyl ester
iFluor® Ultra 647 succinimidyl ester
iFluor® Ultra 750 succinimidyl ester
iFluor® 720 succinimidyl ester
iFluor® 740 succinimidyl ester
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iFluor® 770 succinimidyl ester
iFluor® 780 succinimidyl ester
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OverviewpdfSDSpdfProtocol


Molecular weight
1341.71
Absorbance (nm)
587
Correction Factor (260 nm)
0.05
Correction Factor (280 nm)
0.04
Extinction coefficient (cm -1 M -1)
1800001
Excitation (nm)
588
Emission (nm)
604
Quantum yield
0.531
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. iFluor® 594 Styramide is a superior replacement for Alexa Fluor 594 tyramide or other spectrally similar fluorescent tyramide conjugates or TSA reagents.

Platform


Fluorescence microscope

ExcitationCy3/TRITC filter set
EmissionCy3/TRITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)Cy3/TRITC filter set

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

Cat. #

Product Name

Unit

Ex (nm)

Em (nm)

45000

iFluor™ 350 Styramide

100 slides

345

442

45020

iFluor™ 488 Styramide

100 slides

491

514

45025

iFluor™ 546 Styramide

100 slides

541

557

45027

iFluor™ 555 Styramide

100 slides

552

567

45030

iFluor™ 568 Styramide

100 slides

568

587

45035

iFluor™ 594 Styramide

100 slides

592

619

45045

iFluor™ 647 Styramide

100 slides

649

665

45050

iFluor™ 680 Styramide

100 slides

676

695

45055

iFluor™ 700 Styramide

100 slides

685

710

45065

iFluor™ 750 Styramide

100 slides

749

775

45070

iFluor™ 790 Styramide 100 slides 782 811

45300

Biotin Styramide 100 slides    
45305 DIG Styramide 100 slides    
45310 DNP Styramide 100 slides    

PREPARATION OF STOCK SOLUTION

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. Styramide™ stock solution (100X):
    Add 100 µL of DMSO into the vial of iFluor™ dye-labeled 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 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. 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® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide* 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 mM74.532 µL372.659 µL745.318 µL3.727 mL7.453 mL
5 mM14.906 µL74.532 µL149.064 µL745.318 µL1.491 mL
10 mM7.453 µL37.266 µL74.532 µL372.659 µL745.318 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

Absorbance (nm)587
Correction Factor (260 nm)0.05
Correction Factor (280 nm)0.04
Extinction coefficient (cm -1 M -1)1800001
Excitation (nm)588
Emission (nm)604
Quantum yield0.531

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 594 maleimide58860418000010.5310.050.04
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® 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® 594 Tyramide58860418000010.5310.050.04
iFluor® 594 TCO58860418000010.5310.050.04
iFluor® 594 Tetrazine58860418000010.5310.050.04
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® 610 Styramide61062811000010.8510.320.49
iFluor® 660 Styramide66367825000010.2610.070.08
iFluor® 405 Styramide4034273700010.9110.480.77
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