AAT Bioquest

Fluorescein Tyramide

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Physical properties
Molecular weight495.49
Spectral properties
Absorbance (nm)487
Correction Factor (260 nm)0.32
Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)800001
Excitation (nm)498
Emission (nm)517
Quantum yield0.79001, 0.952
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


Molecular weight
Absorbance (nm)
Correction Factor (260 nm)
Correction Factor (280 nm)
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Quantum yield
0.79001, 0.952
Fluorescein Tyramide is a green fluorescent reagent widely used for tyramide signal amplification (TSA) in IHC, ICC, FISH and multicolor FISH. HRP catalyzes localized deposition of multiple tyramide molecules (catalyzed reporter deposition, CARD), binding the fluorescein tyramide to adjacent tyrosines to enhance fluorescent signal. Under the same conditions, iFluor® 488 Styramide is a superior replacement with higher sensitivity and faster reaction speed. In addition, iFluor 488 is much more photostable, and tolerate much broad pH ranges.


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC 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 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


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fluorescein 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 mM201.82 µL1.009 mL2.018 mL10.091 mL20.182 mL
5 mM40.364 µL201.82 µL403.641 µL2.018 mL4.036 mL
10 mM20.182 µL100.91 µL201.82 µL1.009 mL2.018 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

Absorbance (nm)487
Correction Factor (260 nm)0.32
Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)800001
Excitation (nm)498
Emission (nm)517
Quantum yield0.79001, 0.952

Product Family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Fluorescein biotin4985178000010.79001, 0.9520.320.35
Fluorescein dicaprylate [Fluorescein dioctanoate]4985178000010.79001, 0.9520.320.35
Fluorescein DHPE4985178000010.79001, 0.9520.320.35
Cy3 tyramide55556915000010.1510.070.073
Cy5 tyramide65167025000010.271, 0.420.020.03
Fluorescein aldehyde [5-FAM aldehyde]49351783000-0.320.178
Cy7 tyramide7567792500000.30.050.036
XFD514 tyramide51854380000-0.310.18
XFD532 tyramide534553810000.6110.240.09



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