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AAT Bioquest

XFD546 tyramide reagent *Same Structure to Alexa Fluor™ 546 tyramide*

Product key features

  • Ex/Em: 561/572 nm
  • Extinction coefficient: 112,000 cm-1M-1
  • Tyramide Signal Amplification (TSA): Facilitates ultrasensitive detection of low-abundance targets, ideal for IHC and ICC applications
  • Bright & Stable XFD546 Dye: Delivers high quantum yield with robust resistance to photobleaching, temperature shifts, and pH variations (4–10)

Product description

XFD546 is manufactured by AAT Bioquest, and it has the same chemical structure of Alexa Fluor® 546 (Alexa Fluor® is the trademark of ThermoFisher). For many immunohistochemical (IHC) applications, the traditional enzymatic amplification procedures are sufficient for achieving adequate antigen detection. However, several factors limit the sensitivity and utility of these procedures. 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 translates ultrasensitive detection of low-abundance targets and the use of smaller amounts of antibodies and hybridization probes. In immunohistochemical applications, sensitivity enhancements derived from TSA method allow primary antibody dilutions to be increased to reduce nonspecific background signals, and can overcome weak immunolabeling caused by suboptimal fixation procedures or low levels of target expression. XFD546 tyramide contains the bright XFD546 dye that can be readily detected with the standard TRITC 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

Tyramide stock solution (200X)

Add 100 µL of DMSO to the vial of XFD546 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.

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 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 the 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 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 a 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 a lower concentration of the tyramide reagent 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 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 XFD546 tyramide reagent *Same Structure to Alexa Fluor™ 546 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 mM77.95 µL389.748 µL779.496 µL3.897 mL7.795 mL
5 mM15.59 µL77.95 µL155.899 µL779.496 µL1.559 mL
10 mM7.795 µL38.975 µL77.95 µL389.748 µL779.496 µL

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

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
XFD488 tyramide reagent *Same Structure to Alexa Fluor™ 488 tyramide*499520730000.9210.30.11
XFD594 tyramide reagent *Same Structure to Alexa Fluor™ 594 tyramide*590618920000.6610.430.56
XFD350 tyramide reagent *Same Structure to Alexa Fluor™ 350 tyramide*34344119000-0.250.19
XFD568 tyramide reagent *Same Structure to Alexa Fluor™ 568 tyramide*579603880000.6910.450.46

Citations

View all 16 citations: Citation Explorer
Dynamic regulation of proximal tubular autophagy from injury to repair after ischemic kidney damage
Authors: Gong, Yuhong and Zhu, Wei and Li, Yongqiang and Lu, Tao and Tan, Jiexing and He, Changsheng and Yang, Luodan and Zhu, Yufeng and Gong, Li
Journal: Cellular \& Molecular Biology Letters (2024): 151
Electroretinography Changes in Feline Model of Iodoacetic Acid-induced Retinal Degeneration
Authors: Kim, Changzoo and Lee, Myungshin and Lee, Seung Uk and Lee, Sang Joon
Journal: Journal of the Korean Ophthalmological Society (2019): 1205--1215
An ultrasensitive electrochemical immunosensor for procalcitonin detection based on the gold nanoparticles-enhanced tyramide signal amplification strategy
Authors: Liu, P., Li, C., Zhang, R., Tang, Q., Wei, J., Lu, Y., Shen, P.
Journal: Biosens Bioelectron (2019): 543-550
A amperometric immunosensor for sensitive detection of circulating tumor cells using a tyramide signal amplification-based signal enhancement system
Authors: Zhou, X., Li, Y., Wu, H., Huang, W., Ju, H., Ding, S.
Journal: Biosens Bioelectron (2019): 88-94
Gold nanoparticle labeling with tyramide signal amplification for highly sensitive detection of alpha fetoprotein in human serum by ICP-MS
Authors: Li, X., Chen, B., He, M., Xiao, G., Hu, B.
Journal: Talanta (2018): 40-46

References

View all 74 references: Citation Explorer
Tyramide Signal Amplification for Immunofluorescent Enhancement
Authors: Faget L, Hnasko TS.
Journal: Methods Mol Biol (2015): 161
Enhanced detection of Porcine reproductive and respiratory syndrome virus in fixed tissues by in situ hybridization following tyramide signal amplification
Authors: Trang NT, Hirai T, Ngan PH, Lan NT, Fuke N, Toyama K, Yamamoto T, Yamaguchi R.
Journal: J Vet Diagn Invest (2015): 326
Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification
Authors: Lauter G, Soll I, Hauptmann G.
Journal: Methods Mol Biol (2014): 175
KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells
Authors: Garrigues HJ, Rubinchikova YE, Rose TM.
Journal: Virology (2014): 75
Rapid and sensitive detection of Escherichia coli O157:H7 in milk and ground beef using magnetic bead-based immunoassay coupled with tyramide signal amplification
Authors: Aydin M, Herzig GP, Jeong KC, Dunigan S, Shah P, Ahn S.
Journal: J Food Prot (2014): 100
Page updated on December 11, 2024

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Catalog Number11075
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Physical properties

Molecular weight

1282.88

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.12

Extinction coefficient (cm -1 M -1)

112000

Excitation (nm)

561

Emission (nm)

572

Quantum yield

0.791

Storage, safety and handling

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

ExcitationCy3, TRITC filter set
EmissionCy3, TRITC filter set
Recommended plateBlack wall, clear bottom