AAT Bioquest

Cy5 tyramide

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Physical properties
Molecular weight890.00
Spectral properties
Correction Factor (260 nm)0.02
Correction Factor (280 nm)0.03
Correction Factor (482 nm)0.009
Correction Factor (565 nm)0.09
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)651
Emission (nm)670
Quantum yield0.271, 0.42
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
Direct upgrades
iFluor® 647 Tyramide


Molecular weight
Correction Factor (260 nm)
Correction Factor (280 nm)
Correction Factor (482 nm)
Correction Factor (565 nm)
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Quantum yield
0.271, 0.42
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. Cy5 tyramide contains the bright Cy5 that can be readily detected with the standard Cy5 filter set.


Fluorescence microscope

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

Example protocol


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


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. 


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.


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:


Peroxidase labeling
  1. Optional: Quench endogenous peroxidase activity by incubating cell or tissue sample in a 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 with biotin blocking buffer. 

  3. Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.

  4. Remove the blocking solution and add the primary antibody diluted in the 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 the 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 the 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 instructions 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.


Product Name

Ex/Em (nm)


Nuclear Blue™ DCS1



Nuclear Green™ DCS1



Nuclear Orange™ DCS1



Nuclear Red™ DCS1



Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Cy5 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 mM112.36 µL561.798 µL1.124 mL5.618 mL11.236 mL
5 mM22.472 µL112.36 µL224.719 µL1.124 mL2.247 mL
10 mM11.236 µL56.18 µL112.36 µL561.798 µL1.124 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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Spectral properties

Correction Factor (260 nm)0.02
Correction Factor (280 nm)0.03
Correction Factor (482 nm)0.009
Correction Factor (565 nm)0.09
Extinction coefficient (cm -1 M -1)2500001
Excitation (nm)651
Emission (nm)670
Quantum yield0.271, 0.42

Product Family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)Correction Factor (482 nm)Correction Factor (565 nm)
Cy5 tertrazine [Cy5 tertrazine]65167025000010.271, 0.420.020.030.0090.09
Cy5 phosphoramidite65167025000010.271, 0.420.020.030.0090.09
Cy5 tetrazine65167025000010.271, 0.420.020.030.0090.09
Cy5 aldehyde65167025000010.271, 0.420.020.030.0090.09
DBCO-Cy565167025000010.271, 0.420.020.030.0090.09
Cy3 tyramide55556915000010.1510.070.073--
Cy7 tyramide7567792500000.30.050.0360.00050.0193
XFD514 tyramide51854380000-0.310.18--
XFD532 tyramide534553810000.6110.240.09--
Fluorescein Tyramide4985178000010.79001, 0.9520.320.35--
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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