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iFluor® 450 Tyramide *Superior Replacement for Opal 480*

Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using iFluor<strong>™</strong> 450 Tyramide. Human lung adenocarcinoma positive tissue sections were stained with Mouse anti-EpCAM or Control Mouse IgG antibody and then incubated with polyHRP-labeled Goat anti-Mouse IgG secondary antibody followed by iFluor® 450 Tyramide™ (Cat#45097).
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using iFluor<strong>™</strong> 450 Tyramide. Human lung adenocarcinoma positive tissue sections were stained with Mouse anti-EpCAM or Control Mouse IgG antibody and then incubated with polyHRP-labeled Goat anti-Mouse IgG secondary antibody followed by iFluor® 450 Tyramide™ (Cat#45097).
<strong>Superior sensitivity with iFluor® 450 tyramide.</strong> HeLa cells were incubated with primary anti-tubulin antibodies followed by detection with HRP-Goat anti-Mouse IgG and<strong><em> </em></strong>iFluor® 450 tyramide (Left) or Alexa Fluor® 450 tyramide (Right). Fluorescence images were taken on a Keyence BZ-X710 fluorescence microscope equipped with a FITC filter set.
Ordering information
Price ()
Catalog Number45097
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight625.78
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.45
Correction Factor (280 nm)0.27
Extinction coefficient (cm -1 M -1)400001
Excitation (nm)451
Emission (nm)502
Quantum yield0.821
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
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iFluor® 810 goat anti-rabbit IgG (H+L)
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iFluor® 820 goat anti-rabbit IgG (H+L)
iFluor® 820 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 594 succinimidyl ester
iFluor® 633 succinimidyl ester
iFluor® 647 succinimidyl ester
iFluor® 660 succinimidyl ester
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iFluor® 700 succinimidyl ester
iFluor® 750 succinimidyl ester
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iFluor® 450 succinimidyl ester
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iFluor® 440 succinimidyl ester
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iFluor® Ultra 594 succinimidyl ester
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iFluor® 720 succinimidyl ester
iFluor® 740 succinimidyl ester
iFluor® 597 succinimidyl ester
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OverviewpdfSDSpdfProtocol


Molecular weight
625.78
Correction Factor (260 nm)
0.45
Correction Factor (280 nm)
0.27
Extinction coefficient (cm -1 M -1)
400001
Excitation (nm)
451
Emission (nm)
502
Quantum yield
0.821
iFluor® 450 tyramide is optimized to a superior replacement for Opal 480 or other spectrally similar fluorescent tyramide conjugates or TSA reagents. Tyramide reagetns can be used to detect extremely low-abundance targets in cells and tissues with significantly improved fluorescence signal than the direct fluorescence labeling reagents. In combination with our superior iFluor® dyes that have higher florescence intensity, increased photostability and enhanced water solubility, the iFluor® dye-labeled tyramide conjugates can generate fluorescence signal with significantly higher precision and sensitivity.

Platform


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC filter set

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.

iFluor™ 450 Tyramide stock solution (200X)
Add 100 µL DMSO to vial and mix well.
Note     Unused Tyramide stock solution can be stored at 2-8 °C.

PREPARATION OF WORKING SOLUTION

iFluor™ 450 Tyramide working solution (1X)
Add 100 µL of Tyramide stock solution into 20 mL of buffer of your choice containing 0.003% H2O2.
Note     Tris Buffer, pH=7.4 can be used for optimal performance.
Note     Tyramide working solution should be used immediately and made fresh on the day of use.
Note     20 mL solution is good for 200 tests.

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.
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 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.
  3. Use the appropriate filter set to visualize the signal from the tyramide labeling. 
Table 1.Products recommended for nucleus counterstain.
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® 450 Tyramide *Superior Replacement for Opal 480* 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 mM159.801 µL799.003 µL1.598 mL7.99 mL15.98 mL
5 mM31.96 µL159.801 µL319.601 µL1.598 mL3.196 mL
10 mM15.98 µL79.9 µL159.801 µL799.003 µL1.598 mL

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)0.45
Correction Factor (280 nm)0.27
Extinction coefficient (cm -1 M -1)400001
Excitation (nm)451
Emission (nm)502
Quantum yield0.821

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 488 tyramide4915167500010.910.210.11
iFluor® 450 maleimide4515024000010.8210.450.27
iFluor® 555 Tyramide55757010000010.6410.230.14
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 Tyramide58860418000010.5310.050.04
iFluor® 450 Styramide *Superior Replacement for Opal Polaris 480*4515024000010.8210.450.27
iFluor® 633 tyramide64065425000010.2910.0620.044
iFluor® 430 Tyramide *Superior Replacement for Opal 480*4334984000010.7810.680.3
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References


View all 1 references: Citation Explorer
Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study.
Authors: Taube, Janis M and Roman, Kristin and Engle, Elizabeth L and Wang, Chichung and Ballesteros-Merino, Carmen and Jensen, Shawn M and McGuire, John and Jiang, Mei and Coltharp, Carla and Remeniuk, Bethany and Wistuba, Ignacio and Locke, Darren and Parra, Edwin R and Fox, Bernard A and Rimm, David L and Hoyt, Cliff
Journal: Journal for immunotherapy of cancer (2021)