iFluor® 430 Tyramide Superior Replacement for Opal 480

Name: iFluor® 430 Tyramide *Superior Replacement for Opal 480*
Brand: AAT Bioquest
SKU: 45096
Price: 315 USD
Availability: InStock

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

Fix/permeabilize/block cells or tissue
Add primary antibody in blocking buffer
Add HRP-conjugated secondary antibody
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

Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
Rinse the cells or tissue with PBS twice.
Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
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

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.
Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.
Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.
Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4 °C.
Wash with PBS three times for 5 minutes each.
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.
Wash with PBS three times for 5 minutes each.

Tyramide labeling

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.
Rinse with PBS three times.

Counterstain and fluorescence imaging

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.
Mount the coverslip using a mounting medium with anti-fading properties.
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

Open in Advanced Spectrum Viewer

Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 488 tyramide	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 430 maleimide	433	498	40000¹	0.78¹	0.68	0.3
iFluor® 555 Tyramide	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 647 Tyramide	656	670	250000¹	0.25¹	0.03	0.03
iFluor® 350 Tyramide	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 546 Tyramide	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 568 Tyramide	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 594 Tyramide	587	603	200000¹	0.53¹	0.05	0.04
iFluor® 633 tyramide	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 450 Tyramide Superior Replacement for Opal 480	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 680 Tyramide Superior Replacement for Opal 690	684	701	220000¹	0.23¹	0.097	0.094
iFluor® 430 acid	433	498	40000¹	0.78¹	0.68	0.3
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 November 21, 2024

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Catalog Number	45096
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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 Origin	Download PDF
H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12171501