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iFluor® 647 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® 647 tyramide (Cat No. 45110) and detected with a Cy5 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® 647 tyramide (Cat No. 45110) and detected with a Cy5 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® 647 tyramide (Cat No. 45110) and detected with a Cy5 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
<strong>Superior sensitivity with iFluor® 647 tyramide.</strong>&nbsp;HeLa cells were incubated with primary anti-tubulin antibodies followed by detection with HRP-Goat anti-Mouse&nbsp;IgG and&nbsp;iFluor® 647 tyramide (Left) or Alexa Fluor&reg; 647 tyramide (Right). Fluorescence images were taken on a Keyence BZ-X710 fluorescence microscope equipped with a Cy5 filter set.
Gallery Image 3
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Physical properties
Molecular weight1092.20
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251
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® 647 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
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iFluor® 840 goat anti-mouse IgG (H+L)
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iFluor® 860 goat anti-mouse IgG (H+L) *Cross Adsorbed*
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iFluor® 810 goat anti-rabbit IgG (H+L)
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iFluor® 820 goat anti-rabbit IgG (H+L)
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iFluor® 840 goat anti-rabbit IgG (H+L)
iFluor® 840 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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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
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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
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iFluor® 350-Wheat Germ Agglutinin (WGA) Conjugate
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Show More (263)

OverviewpdfSDSpdfProtocol


Molecular weight
1092.20
Correction Factor (260 nm)
0.03
Correction Factor (280 nm)
0.03
Correction Factor (656 nm)
0.0793
Extinction coefficient (cm -1 M -1)
2500001
Excitation (nm)
656
Emission (nm)
670
Quantum yield
0.251
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® 647 tyramide contains the bright iFluor® 647 that can be readily detected with the standard Cy5 filter set. It is an excellent replacement for Alexa Fluor® 647 tyramide (Alexa Fluor® is the trademark of ThermoFisher), Cy5 tyramide, or other comparable fluorescent tyramide conjugates.

Platform


Fluorescence microscope

ExcitationCy5 filter set
EmissionCy5 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 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 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. 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 using a 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 non-specific signal, you can shorten the incubation time with Tyramide. You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use lower concentration of Tyramide 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 counterstaining.

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® 647 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 mM91.558 µL457.792 µL915.583 µL4.578 mL9.156 mL
5 mM18.312 µL91.558 µL183.117 µL915.583 µL1.831 mL
10 mM9.156 µL45.779 µL91.558 µL457.792 µL915.583 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)0.03
Correction Factor (280 nm)0.03
Correction Factor (656 nm)0.0793
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)656
Emission (nm)670
Quantum yield0.251

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 647 maleimide65667025000010.2510.030.03
iFluor® 647 amine65667025000010.2510.030.03
iFluor® 647 hydrazide65667025000010.2510.030.03
iFluor® 647 alkyne65667025000010.2510.030.03
iFluor® 647 azide65667025000010.2510.030.03
iFluor® 488 tyramide4915167500010.910.210.11
iFluor® 647 Styramide *Superior Replacement for Alexa Fluor 647 tyramide*65667025000010.2510.030.03
iFluor® 555 Tyramide55757010000010.6410.230.14
iFluor® 350 Tyramide3454502000010.9510.830.23
iFluor® 546 Tyramide54155710000010.6710.250.15
iFluor® 568 Tyramide56858710000010.5710.340.15
iFluor® 594 Tyramide58760320000010.5310.050.04
iFluor® 647 TCO65667025000010.2510.030.03
iFluor® 647 Tetrazine65667025000010.2510.030.03
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
Show More (9)

Images


Citations


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Immune-regulating bimetallic metal-organic framework nanoparticles designed for cancer immunotherapy
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Authors: Gerlach, Brennan D and Ampomah, Patrick B and Yurdagul Jr, Arif and Liu, Chuang and Lauring, Max C and Wang, Xiaobo and Kasikara, Canan and Kong, Na and Shi, Jinjun and Tao, Wei and others,
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Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage
Authors: Ilnytska, Olga and Lai, Kimberly and Gorshkov, Kirill and Schultz, Mark L and Tran, Bruce Nguyen and Jeziorek, Maciej and Kunkel, Thaddeus J and Azaria, Ruth D and McLoughlin, Hayley S and Waghalter, Miriam and others,
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Pharmacological targeting of Sam68 functions in colorectal cancer stem cells
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References


View all 4 references: Citation Explorer
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
Dual enhancement of triple immunofluorescence using two antibodies from the same species.
Authors: Nakamura, Ayako and Uchihara, Toshiki
Journal: Journal of neuroscience methods (2004): 67-70
Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry.
Authors: Pougnard, Claire and Catala, Philippe and Drocourt, Jean-Louis and Legastelois, Stephane and Pernin, Pierre and Pringuez, Emmanuelle and Lebaron, Philippe
Journal: Applied and environmental microbiology (2002): 3102-7
Oligonucleotides as hybridization probes to localize phytoplasmas in host plants and insect vectors.
Authors: Webb, D R and Bonfiglioli, R G and Carraro, L and Osler, R and Symons, R H
Journal: Phytopathology (1999): 894-901