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iFluor® 555 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® 555 tyramide (Cat No. 45105) and detected with a TRITC/Cy3 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® 555 tyramide (Cat No. 45105) and detected with a TRITC/Cy3 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® 555 tyramide (Cat No. 45105) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
<strong>Superior sensitivity with iFluor® 555 tyramide.</strong> HeLa cells were incubated with primary anti-tubulin antibodies followed by detection with HRP-Goat anti-Mouse IgG and&nbsp;iFluor® 555 tyramide (Left) or Alexa Fluor&reg; 555 tyramide (Right). Fluorescence images were taken on a Keyence BZ-X710 fluorescence microscope equipped with a Cy3 filter set.
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Telephone1-800-990-8053
Fax1-800-609-2943
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Physical properties
Molecular weight1046.41
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.23
Correction Factor (280 nm)0.14
Extinction coefficient (cm -1 M -1)1000001
Excitation (nm)557
Emission (nm)570
Quantum yield0.641
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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OverviewpdfSDSpdfProtocol


Molecular weight
1046.41
Correction Factor (260 nm)
0.23
Correction Factor (280 nm)
0.14
Extinction coefficient (cm -1 M -1)
1000001
Excitation (nm)
557
Emission (nm)
570
Quantum yield
0.641
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® 555 tyramide contains the bright iFluor® 555 that can be readily detected with the standard Cy3 filter set. It is an excellent replacement for Alexa Fluor® 555 tyramide (Alexa Fluor® is the trademark of ThermoFisher), Cy3 tyramide, or other comparable fluorescent tyramide conjugates.

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 (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® 555 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 mM95.565 µL477.824 µL955.648 µL4.778 mL9.556 mL
5 mM19.113 µL95.565 µL191.13 µL955.648 µL1.911 mL
10 mM9.556 µL47.782 µL95.565 µL477.824 µL955.648 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)0.23
Correction Factor (280 nm)0.14
Extinction coefficient (cm -1 M -1)1000001
Excitation (nm)557
Emission (nm)570
Quantum yield0.641

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 555 maleimide55757010000010.6410.230.14
iFluor® 555 amine55757010000010.6410.230.14
iFluor® 555 hydrazide55757010000010.6410.230.14
iFluor® 488 tyramide4915167500010.910.210.11
iFluor® 555 alkyne55757010000010.6410.230.14
iFluor® 555 azide55757010000010.6410.230.14
iFluor® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570*55757010000010.6410.230.14
iFluor® 647 Tyramide65667025000010.2510.030.03
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® 555 TCO55757010000010.6410.230.14
iFluor® 555 Tetrazine55757010000010.6410.230.14
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
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