logo
AAT Bioquest

iFluor® 430 Tyramide *Superior Replacement for Opal 480*

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.

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

Spectrum

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 Tyramide58760320000010.5310.050.04
iFluor® 633 tyramide64065425000010.2910.0620.044
iFluor® 450 Tyramide *Superior Replacement for Opal 480*4515024000010.8210.450.27
iFluor® 680 Tyramide *Superior Replacement for Opal 690*68470122000010.2310.0970.094
iFluor® 430 acid4334984000010.7810.680.3
Show More (3)

Citations

View all 3 citations: Citation Explorer
Membrane progesterone receptor $\gamma$ (paqr5b) is essential for the formation of neurons in the zebrafish olfactory rosette
Authors: Mustary, Umme Habiba and Maeno, Akiteru and Rahaman, Md Mostafizur and Ali, Md Hasan and Tokumoto, Toshinobu
Journal: Scientific Reports (2024): 24354
Intratumoral injection of interferon gamma promotes the efficacy anti-PD1 treatment in colorectal cancer
Authors: Tang, Yang and Wei, Jingsun and Ge, Xiaoxu and Yu, Chengxuan and Lu, Wei and Qian, Yucheng and Yang, Hang and Fu, Dongliang and Fang, Yimin and Zhou, Yinyi and others,
Journal: Cancer Letters (2024): 216798
MDIG-mediated H3K9me3 demethylation upregulates Myc by activating OTX2 and facilitates liver regeneration
Authors: Du, Jinpeng and Liao, Wenwei and Wang, Haichuan and Hou, Guimin and Liao, Min and Xu, Lin and Huang, Jiwei and Yuan, Kefei and Chen, Xiangzheng and Zeng, Yong
Journal: Signal Transduction and Targeted Therapy (2023): 351

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
Page updated on December 6, 2024

Ordering information

Price
Unit size
Catalog Number45096
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

710.89

Solvent

DMSO

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 yield

0.781

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

Platform

Fluorescence microscope

ExcitationViolet filter set
EmissionViolet filter set
Recommended plateBlack wall, clear bottom
<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&nbsp;IgG and&nbsp;iFluor® 430 tyramide&trade; (Left) or Alexa Fluor&reg; 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&nbsp;IgG and&nbsp;iFluor® 430 tyramide&trade; (Left) or Alexa Fluor&reg; 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&nbsp;IgG and&nbsp;iFluor® 430 tyramide&trade; (Left) or Alexa Fluor&reg; 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.