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iFluor® 430 Tyramide *Superior Replacement for Opal 480*

<strong>Superior sensitivity with iFluor® 430 tyramide.</strong> HeLa cells were incubated with primary anti-tubulin antibodies followed by detection with HRP-Goat anti-Mouse IgG and iFluor® 430 tyramide™ (Left) or Alexa Fluor® 430 tyramide (Right). Fluorescence images were taken on a Keyence BZ-X710 fluorescence microscope equipped with a violet filter set.
<strong>Superior sensitivity with iFluor® 430 tyramide.</strong> HeLa cells were incubated with primary anti-tubulin antibodies followed by detection with HRP-Goat anti-Mouse IgG and iFluor® 430 tyramide™ (Left) or Alexa Fluor® 430 tyramide (Right). Fluorescence images were taken on a Keyence BZ-X710 fluorescence microscope equipped with a violet filter set.
Formalin-fixed, paraffin-embedded (FFPE) human lung adenocarcinoma tissue was incubated with an anti-EpCAM primary antibody, and an HRP conjugated anti-mouse secondary antibody. TSA signal was developed by incubation of tissue section with 5 µg/mL of iFluor® 430 tyramide (Cat No. 45096) for 10 minutes. Images were acquired on a confocal microscope equipped with a Violet filter set.
Ordering information
Price ()
Catalog Number45096
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 weight710.89
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.68
Correction Factor (280 nm)0.3
Extinction coefficient (cm -1 M -1)400001
Excitation (nm)433
Emission (nm)498
Quantum yield0.781
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) *Cross Adsorbed*
iFluor® 820 goat anti-rabbit IgG (H+L)
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iFluor® 840 goat anti-rabbit IgG (H+L)
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iFluor® 594 succinimidyl ester
iFluor® 633 succinimidyl ester
iFluor® 647 succinimidyl ester
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iFluor® 670 succinimidyl ester
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OverviewpdfSDSpdfProtocol


Molecular weight
710.89
Correction Factor (260 nm)
0.68
Correction Factor (280 nm)
0.3
Extinction coefficient (cm -1 M -1)
400001
Excitation (nm)
433
Emission (nm)
498
Quantum yield
0.781
iFluor® 430 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

ExcitationViolet filter set
EmissionViolet filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)Violet 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™ 430 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™ 430 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® 430 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 mM140.669 µL703.344 µL1.407 mL7.033 mL14.067 mL
5 mM28.134 µL140.669 µL281.337 µL1.407 mL2.813 mL
10 mM14.067 µL70.334 µL140.669 µL703.344 µL1.407 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.68
Correction Factor (280 nm)0.3
Extinction coefficient (cm -1 M -1)400001
Excitation (nm)433
Emission (nm)498
Quantum yield0.781

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® 430 maleimide4334984000010.7810.680.3
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® 633 tyramide64065425000010.2910.0620.044
iFluor® 450 Tyramide *Superior Replacement for Opal 480*4515024000010.8210.450.27
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References


View all 50 references: Citation Explorer
Immunofluorescent Staining of Adult Murine Paraffin-Embedded Skeletal Tissue.
Authors: Felsenthal, Neta and Zelzer, Elazar
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 337-344
Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide.
Authors: Xiao, Lu and Labaer, Joshua and Guo, Jia
Journal: Cells (2021)
Single-cell RNA sequencing of human liver reveals hepatic stellate cell heterogeneity.
Authors: Payen, Valéry L and Lavergne, Arnaud and Alevra Sarika, Niki and Colonval, Megan and Karim, Latifa and Deckers, Manon and Najimi, Mustapha and Coppieters, Wouter and Charloteaux, Benoît and Sokal, Etienne M and El Taghdouini, Adil
Journal: JHEP reports : innovation in hepatology (2021): 100278
Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide.
Authors: Pham, Thai and Liao, Renjie and Labaer, Joshua and Guo, Jia
Journal: Molecules (Basel, Switzerland) (2021)
Accessibility-dependent topology studies of membrane proteins using a SpyTag/SpyCatcher protein-ligation system.
Authors: Bae, Yoonji and Lee, Sang Kwon and Chae, Young Chan and Park, Chan Young and Kang, Sebyung
Journal: International journal of biological macromolecules (2021): 171-178
Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.
Authors: Jessop, Peter and Gering, Martin
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 193-208
Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.
Authors: Wilbanks, Brandon and Garcia, Brian and Byrne, Shane and Dedon, Peter and Maher, L James
Journal: Chembiochem : a European journal of chemical biology (2021): 1400-1404
Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells.
Authors: Sommaggio, Roberta and Cappuzzello, Elisa and Dalla Pietà, Anna and Tosi, Anna and Palmerini, Pierangela and Carpanese, Debora and Nicolè, Lorenzo and Rosato, Antonio
Journal: Oncoimmunology (2020): 1777046
A simple, real-time assay of horseradish peroxidase using biolayer interferometry.
Authors: Kojima, Takaaki and Nakane, Ayako and Zhu, Bo and Alfi, Almasul and Nakano, Hideo
Journal: Bioscience, biotechnology, and biochemistry (2019): 1822-1828
The EMARS Reaction for Proximity Labeling.
Authors: Honke, Koichi and Miyagawa-Yamaguchi, Arisa and Kotani, Norihiro
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 1-12