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Cy3 tyramide

Immunofluorescent image of paraffin-embedded human lung carcinoma labeled with EpCAM Rabbit mAb followed with HRP-labeled goat anti-rabbit IgG (H+L) (Cat#16793). The signal was developed with AAT’s Cy3 tyramide (Cat#11065, Red). Cells were also counterstained with DAPI (Blue).
Immunofluorescent image of paraffin-embedded human lung carcinoma labeled with EpCAM Rabbit mAb followed with HRP-labeled goat anti-rabbit IgG (H+L) (Cat#16793). The signal was developed with AAT’s Cy3 tyramide (Cat#11065, Red). Cells were also counterstained with DAPI (Blue).
Immunofluorescent image of paraffin-embedded human lung carcinoma labeled with EpCAM Rabbit mAb followed with HRP-labeled goat anti-rabbit IgG (H+L) (Cat#16793). The signal was developed with AAT’s Cy3 tyramide (Cat#11065, Red). Cells were also counterstained with DAPI (Blue).
Gallery Image 2
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Physical properties
Molecular weight863.96
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.073
Extinction coefficient (cm -1 M -1)1500001
Excitation (nm)555
Emission (nm)569
Quantum yield0.151
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
Direct upgrades
iFluor® 555 Tyramide

OverviewpdfSDSpdfProtocol


Molecular weight
863.96
Correction Factor (260 nm)
0.07
Correction Factor (280 nm)
0.073
Extinction coefficient (cm -1 M -1)
1500001
Excitation (nm)
555
Emission (nm)
569
Quantum yield
0.151
For many immunohistochemical (IHC) applications, the traditional enzymatic amplification procedures are sufficient for achieving adequate antigen detection. However, several factors limit the sensitivity and utility of these procedures. 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 translates ultrasensitive detection of low-abundance targets and the use of smaller amounts of antibodies and hybridization probes. In immunohistochemical applications, sensitivity enhancements derived from TSA method allow primary antibody dilutions to be increased to reduce nonspecific background signals, and can overcome weak immunolabeling caused by suboptimal fixation procedures or low levels of target expression. Cy3 tyramide contains the bright Cy3 that can be readily detected with the standard Cy3 filter set.

Platform


Fluorescence microscope

ExcitationCy3/TRITC filter set
EmissionCy3/TRITC 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 (1000X)

Add an appropriate amount of DMSO to make a 1-5 mM tyramide stock solution.

Note: Make single-use aliquots and store unused 1000X stock solution at 2-8 °C, protected from light. Avoid repeat freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

Tyramide working solution (1X)

Add 1 µL of the tyramide stock solution into 1 mL of a buffer of your choice containing 0.003% H2O2.

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

Note: 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 Tyramide. 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 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 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 Cy3 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 mM115.746 µL578.73 µL1.157 mL5.787 mL11.575 mL
5 mM23.149 µL115.746 µL231.492 µL1.157 mL2.315 mL
10 mM11.575 µL57.873 µL115.746 µL578.73 µL1.157 mL

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


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spectrum

Spectral properties

Correction Factor (260 nm)0.07
Correction Factor (280 nm)0.073
Extinction coefficient (cm -1 M -1)1500001
Excitation (nm)555
Emission (nm)569
Quantum yield0.151

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Cy3 tetrazine55556915000010.1510.070.073
Cy3 phosphoramidite55556915000010.1510.070.073
Cy3 aldehyde55556915000010.1510.070.073
DBCO-Cy355556915000010.1510.070.073
Cy5 tyramide65167025000010.271, 0.420.020.03
Cy7 tyramide7567792500000.30.050.036
XFD514 tyramide51854380000-0.310.18
XFD532 tyramide534553810000.6110.240.09
Fluorescein Tyramide4985178000010.79001, 0.9520.320.35

Images


Citations


View all 4 citations: Citation Explorer
FGF2 promotes the expansion of parietal mesothelial progenitor pools and inhibits BMP4-mediated smooth muscle cell differentiation
Authors: Hwang, Youngmin and Shimamura, Yuko and Tanaka, Junichi and Miura, Akihiro and Sawada, Anri and Sarmah, Hemanta and Shimizu, Dai and Kondo, Yuri and Ninish, Zurab and Yamada, Kazuhiko and others,
Journal: bioRxiv (2024): 2024--01
Dynamic expression of mucins and the genes controlling mucin-type O-glycosylation within the mouse respiratory system
Authors: Tian, E and Syed, Zulfeqhar A and Edin, Matthew L and Zeldin, Darryl C and Ten Hagen, Kelly G
Journal: Glycobiology (2023): cwad031
Stage-specific requirement for METTL3-dependent m6A modification during dental pulp stem cell differentiation
Authors: Luo, Haiyun and Liu, Wenjing and Zhou, Yachuan and Zhang, Yanli and Wu, Junrong and Wang, Ruolan and Shao, Longquan
Journal: Journal of Translational Medicine (2022): 1--15
Transient activation of the Notch-her15. 1 axis plays an important role in the maturation of V2b interneurons
Authors: Mizoguchi, Takamasa and Fukada, Michi and Iihama, Miku and Song, Xuehui and Fukagawa, Shun and Kuwabara, Shuhei and Omaru, Shuhei and Higashijima, Shin-ichi and Itoh, Motoyuki
Journal: Development (2020): dev191312

References


View all 74 references: Citation Explorer
Tyramide Signal Amplification for Immunofluorescent Enhancement
Authors: Faget L, Hnasko TS.
Journal: Methods Mol Biol (2015): 161
Enhanced detection of Porcine reproductive and respiratory syndrome virus in fixed tissues by in situ hybridization following tyramide signal amplification
Authors: Trang NT, Hirai T, Ngan PH, Lan NT, Fuke N, Toyama K, Yamamoto T, Yamaguchi R.
Journal: J Vet Diagn Invest (2015): 326
Rapid and sensitive detection of Escherichia coli O157:H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification
Authors: Aydin M, Herzig GP, Jeong KC, Dunigan S, Shah P, Ahn S.
Journal: J Food Prot (2014): 100
Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis
Authors: Stack EC, Wang C, Roman KA, Hoyt CC.
Journal: Methods (2014): 46
KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells
Authors: Garrigues HJ, Rubinchikova YE, Rose TM.
Journal: Virology (2014): 75
Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification
Authors: Lauter G, Soll I, Hauptmann G.
Journal: Methods Mol Biol (2014): 175
Characterization of GABAergic neurons in the mouse lateral septum: a double fluorescence in situ hybridization and immunohistochemical study using tyramide signal amplification
Authors: Zhao C, Eisinger B, Gammie SC.
Journal: PLoS One (2013): e73750
Quantification of alpha-tubulin isotypes by sandwich ELISA with signal amplification through biotinyl-tyramide or immuno-PCR
Authors: Draberova E, Stegurova L, Sulimenko V, Hajkova Z, Draber P.
Journal: J Immunol Methods (2013): 63
Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: endogenous streptavidin-binding sites lead to false positive staining
Authors: Horling L, Neuhuber WL, Raab M.
Journal: J Neurosci Methods (2012): 124
Integrated tyramide and polymerization-assisted signal amplification for a highly-sensitive immunoassay
Authors: Yuan L, Xu L, Liu S.
Journal: Anal Chem (2012): 10737