logo
AAT Bioquest

iFluor® 647 Tyramide

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.

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

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
iFluor® 647 acid65667025000010.2510.030.03
Show More (10)

Citations

View all 11 citations: Citation Explorer
H3K9 lactylation in malignant cells facilitates CD8+ T cell dysfunction and poor immunotherapy response
Authors: Wang, Ruijie and Li, Chuwen and Cheng, Zhongyi and Li, Mingyu and Shi, Jianbo and Zhang, Zhiyuan and Jin, Shufang and Ma, Hailong
Journal: Cell Reports (2024)
The tandem CD33-CLL1 CAR-T as an approach to treat acute myeloid leukemia: The tandem CLL1/CD33 CAR-T to treat AML
Authors: Wang, Huiru and Feng, Shanglong and Zhu, Yanliang and Zhang, Yafeng and Zhou, Ziwei and Nian, Zhigang and Lu, Xueqin and Peng, Peng and Wu, Shu and Zhou, Li
Journal: Blood Transfusion (2024)
Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis
Authors: Hu, Xingxing and Zou, Menglin and Zheng, Weishuai and Zhu, Minghui and Hou, Qinhui and Gao, Han and Zhang, Xin and Liu, Yuan and Cheng, Zhenshun
Journal: Respiratory Research (2024): 1--13
Digital Light Processing 3D Printing of Gyroid Scaffold with Isosorbide-Based Photopolymer for Bone Tissue Engineering
Authors: Verisqa, Fiona and Cha, Jae-Ryung and Nguyen, Linh and Kim, Hae-Won and Knowles, Jonathan C
Journal: Biomolecules (2022): 1692
Immune-regulating bimetallic metal-organic framework nanoparticles designed for cancer immunotherapy
Authors: Dai, Zan and Wang, Qiaoyun and Tang, Jie and Wu, Min and Li, Haoze and Yang, Yannan and Zhen, Xu and Yu, Chengzhong
Journal: Biomaterials (2022): 121261

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
Page updated on October 11, 2024

Ordering information

Price
Unit size
Catalog Number45110
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

1092.20

Solvent

DMSO

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 yield

0.251

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationCy5 filter set
EmissionCy5 filter set
Recommended plateBlack wall, clear bottom
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