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iFluor® 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 tyramide (Cat No. 45107) 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 tyramide (Cat No. 45107) 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 tyramide (Cat No. 45107) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
<strong>Superior sensitivity with iFluor® 594 tyramide.</strong> HeLa cells were incubated with primary anti-tubulin antibodies followed by detection with HRP-Goat anti-Mouse&nbsp;IgG and&nbsp;iFluor® 594 tyramide (Left) or Alexa Fluor&reg; 594 tyramide (Right). Fluorescence images were taken on a Keyence BZ-X710 fluorescence microscope equipped with a TRITC filter set.
Gallery Image 3
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Physical properties
Molecular weight841.95
SolventDMSO
Spectral properties
Absorbance (nm)587
Correction Factor (260 nm)0.05
Correction Factor (280 nm)0.04
Extinction coefficient (cm -1 M -1)2000001
Excitation (nm)587
Emission (nm)603
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
UNSPSC12171501
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iFluor® 860 goat anti-mouse IgG (H+L)
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iFluor® 810 goat anti-rabbit IgG (H+L)
iFluor® 810 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 820 goat anti-rabbit IgG (H+L)
iFluor® 820 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 840 goat anti-rabbit IgG (H+L)
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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
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iFluor® 810 succinimidyl ester
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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
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iFluor® 350-Wheat Germ Agglutinin (WGA) Conjugate
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Show More (267)

OverviewpdfSDSpdfProtocol


Molecular weight
841.95
Absorbance (nm)
587
Correction Factor (260 nm)
0.05
Correction Factor (280 nm)
0.04
Extinction coefficient (cm -1 M -1)
2000001
Excitation (nm)
587
Emission (nm)
603
Quantum yield
0.531
For many immunohistochemical (IHC) applications, traditional enzymatic amplification procedures are sufficient for achieving adequate antigen detection. However, several factors limit their sensitivity and utility. Tyramide signal amplification (TSA) has proven to be a particularly versatile and powerful enzyme amplification technique with improved assay sensitivity. TSA is based on the ability of HRP, in the presence of low concentrations of hydrogen peroxide, to convert labeled tyramine-containing substrate into an oxidized, highly reactive free radical that can covalently bind to tyrosine residues at or near the HRP. To achieve maximal IHC detection, tyramine is prelabeled with a fluorophore. The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label results in the ability to detect low-abundance targets with ultrasensitive precision and reduces the amount of antibodies and hybridization probes needed. In IHC applications, this method can also enhance sensitivity in cases where the primary antibody dilution needs to be increased to reduce nonspecific background signals or overcome weak immunolabeling due to suboptimal fixation procedures or low levels of target expression. The iFluor® 594 tyramide contains the bright iFluor® 594 that can be readily detected with the standard Texas Red filter set, as well as with a TRITC or Cy3 filter set. iFluor® dyes have higher fluorescence intensity, increased photostability, and enhanced water solubility, resulting in fluorescence signals with significantly higher precision and sensitivity. iFluor® 594 is an excellent replacement for Alexa Fluor® 594 tyramide (Alexa Fluor® is the trademark of ThermoFisher) or other comparable fluorescent tyramide conjugates.

Platform


Fluorescence microscope

ExcitationCy3/TRITC filter set
EmissionCy3/TRITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)Compatible with Texas Red 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 tyramide 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

Tyramide stock solution (200X)

Add 100 µL of DMSO to the vial of iFluor® tyramide and mix well.

Note: Make single-use aliquots and store unused 200X stock solution at 2-8 °C, protected from light. Avoid repeat freeze-thaw cycles. 

PREPARATION OF WORKING SOLUTION

Tyramide working solution (1X)

Add 100 µL of the tyramide stock solution into 20 mL of a buffer of your choice containing 0.003% H2O2.

Note: For optimal performance, use Tris Buffer, pH=7.4.

Note: A 20 mL solution is good for 200 tests. The tyramide working solution should be used immediately and made fresh on the day of use. Avoid direct exposure to light.

Secondary antibody-HRP working solution

Make an appropriate concentration of secondary antibody-HRP working solution 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 the preferred specific solution/protocol as needed. A 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.
Tyramide labeling
  1. Prepare and apply 100 µL of Tyramide working solution to each sample and incubate for 5-10 minutes at room temperature.

    Note: If you observe a non-specific signal, you can shorten the incubation time with the tyramide reagent. You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use a lower concentration of the tyramide reagent 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.

    Note: To ensure optimal results, it is recommended to use either ReadiUse™ microscope mounting solution (Cat. 20009) or FluoroQuest™ TSA/PSA Antifade Mounting Medium *Optimized for Tyramide and Styramide Imaging* (Cat. 44890) instead of Vectashield® mounting media. There are instances where Vectashield® mounting media may not be suitable for certain TSA/PSA conjugates.

  3. Use the appropriate filter set to visualize the signal from the Tyramide labeling.

Table 1. Products recommended for nucleus counterstain

Cat#Product NameEx/Em (nm)
17548Nuclear Blue™ DCS1350/461
17550Nuclear Green™ DCS1503/526
17551Nuclear Orange™ DCS1528/576
17552Nuclear Red™ DCS1642/660

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 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 mM118.772 µL593.859 µL1.188 mL5.939 mL11.877 mL
5 mM23.754 µL118.772 µL237.544 µL1.188 mL2.375 mL
10 mM11.877 µL59.386 µL118.772 µL593.859 µL1.188 mL

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)2000001
Excitation (nm)587
Emission (nm)603
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® 488 tyramide4915167500010.910.210.11
iFluor® 594 maleimide58760320000010.5310.050.04
iFluor® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide*58760320000010.5310.050.04
iFluor® 555 Tyramide55757010000010.6410.230.14
iFluor® 647 Tyramide65667025000010.2510.030.03
iFluor® 350 Tyramide3454502000010.9510.830.23
iFluor® 546 Tyramide54155710000010.6710.250.15
iFluor® 568 Tyramide56858710000010.5710.340.15
iFluor® 594 TCO58760320000010.5310.050.04
iFluor® 594 Tetrazine58760320000010.5310.050.04
iFluor® 633 tyramide64065425000010.2910.0620.044
iFluor® 430 Tyramide *Superior Replacement for Opal 480*4334984000010.7810.680.3
iFluor® 450 Tyramide *Superior Replacement for Opal 480*4515024000010.8210.450.27
iFluor® 680 Tyramide *Superior Replacement for Opal 690*68470122000010.2310.0970.094
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Images


References


View all 14 references: Citation Explorer
Time Gated Luminescence Imaging of Immunolabeled Human Tissues.
Authors: Chen, Ting and Hong, Rui and Magda, Darren and Bieniarz, Christopher and Morrison, Larry and Miller, Lawrence W
Journal: Analytical chemistry (2017): 12713-12719
Whole Mount RNA-FISH on Ovules and Developing Seeds.
Authors: Bleckmann, Andrea and Dresselhaus, Thomas
Journal: Methods in molecular biology (Clifton, N.J.) (2017): 159-171
Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis.
Authors: Chung, Chen-yo and Cook, Charles E and Lin, Gee-way and Huang, Ting-Yu and Chang, Chun-che
Journal: Insect science (2014): 265-77
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
NIR-labeled perfluoropolyether nanoemulsions for drug delivery and imaging.
Authors: O'Hanlon, Claire E and Amede, Konjit G and O'Hear, Meredith R and Janjic, Jelena M
Journal: Journal of fluorine chemistry (2012): 27-33
Anisotropic magnetic porous assemblies of oxide nanoparticles interconnected via silica bridges for catalytic application.
Authors: Wacker, Josias B and Parashar, Virendra K and Gijs, Martin A M
Journal: Langmuir : the ACS journal of surfaces and colloids (2011): 4380-5
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
mRNA-targeted fluorescent in situ hybridization (FISH) of Gram-negative bacteria without template amplification or tyramide signal amplification.
Authors: Coleman, James R and Culley, David E and Chrisler, William B and Brockman, Fred J
Journal: Journal of microbiological methods (2007): 246-55
Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification.
Authors: Clay, Hilary and Ramakrishnan, Lalita
Journal: Zebrafish (2005): 105-11
Oligo(dA-dT)-dependent signal amplification for the detection of proteins in cells.
Authors: Hanaki, Ken-ichi and Ohka, Seii and Yamamoto, Kenji and Nomoto, Akio and Yoshikura, Hiroshi
Journal: BioTechniques (2004): 856-60, 862-3