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iFluor® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570*

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® 555 styramide (Cat No. 45027) 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® 555 styramide (Cat No. 45027) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
Fluorescence IHC for HER2/ErbB2 detection in formaldehyde-fixed paraffin-embedded breast cancer tissue. Human Her2/Neu (c-erbB-2) positive tissue sections were incubated with Rabbit mAb HER2/ErbB2 diluted 1:5000 (Top) or 1:250 (Bottom), respectively. The tissue was then incubated with HRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 555 Styramide™ (Top) or Alexa Fluor® 555 tyramide (Bottom), respectively. Compared to tyramide amplification, the increase in sensitivity of Styramide™ super signal amplification allows the use of fewer primary antibodies. The same level of sensitivity can be achieved with a significant reduction in the amount of target antibody used (10-50 fold). The cell nucleus was costained with DAPI (Cat No. 17507).
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue  using PSA<strong> ™ </strong>and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with rabbit anti-EpCam antibody and then incubated with polyHRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 555 Styramide™ (Cat#45027) or Alexa Fluor® 555 tyramide stain respectively.  Images showed that iFluor 555™ PSA™ super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor® 555 TSA method.
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 Number45027
Unit Size
Find Distributor
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 weight1023.15
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.23
Correction Factor (280 nm)0.14
Extinction coefficient (cm -1 M -1)1000001
Excitation (nm)557
Emission (nm)570
Quantum yield0.641
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® 790 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 350 goat anti-rabbit IgG (H+L)
iFluor® 405 goat anti-rabbit IgG (H+L)
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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)
iFluor® 350 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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*
iFluor® 532 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 860-streptavidin conjugate
iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate
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iFluor® 594-Wheat Germ Agglutinin (WGA) Conjugate
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iFluor® 430 Tyramide *Superior Replacement for Opal 480*
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iFluor® 770 maleimide
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iFluor® 405 succinimidyl ester
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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
iFluor® 597 succinimidyl ester
iFluor® 770 succinimidyl ester
iFluor® 780 succinimidyl ester
iFluor® 570 succinimidyl ester
iFluor® 830 acid
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Show More (238)

OverviewpdfSDSpdfProtocol


Molecular weight
1023.15
Correction Factor (260 nm)
0.23
Correction Factor (280 nm)
0.14
Extinction coefficient (cm -1 M -1)
1000001
Excitation (nm)
557
Emission (nm)
570
Quantum yield
0.641
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® 555 Styramide is a superior replacement for Alexa Fluor 555 tyramide, Opal 570 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 

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. 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 °C 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-tissueimmunohistochemistry-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® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570* 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 mM97.737 µL488.687 µL977.374 µL4.887 mL9.774 mL
5 mM19.547 µL97.737 µL195.475 µL977.374 µL1.955 mL
10 mM9.774 µL48.869 µL97.737 µL488.687 µL977.374 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.23
Correction Factor (280 nm)0.14
Extinction coefficient (cm -1 M -1)1000001
Excitation (nm)557
Emission (nm)570
Quantum yield0.641

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 555 maleimide55757010000010.6410.230.14
iFluor® 555 amine55757010000010.6410.230.14
iFluor® 555 hydrazide55757010000010.6410.230.14
iFluor® 555 alkyne55757010000010.6410.230.14
iFluor® 555 azide55757010000010.6410.230.14
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® 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® 555 Tyramide55757010000010.6410.230.14
iFluor® 555 TCO55757010000010.6410.230.14
iFluor® 555 Tetrazine55757010000010.6410.230.14
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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