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AAT Bioquest

XFD488 tyramide reagent *Same Structure to Alexa Fluor™ 488 tyramide*

Product key features

  • Ex/Em: 499/520 nm
  • Extinction coefficient: 71,000 cm-1M-1
  • Tyramide Signal Amplification (TSA): Facilitates ultrasensitive detection of low-abundance targets, ideal for IHC and ICC applications
  • Bright & Stable XFD488 Dye: Delivers high quantum yield with robust resistance to photobleaching and pH variations (4–10)

Product description

XFD488 is manufactured by AAT Bioquest, and it has the same chemical structure of Alexa Fluor® 488 (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. XFD488 tyramide contains the bright XFD488 dye that can be readily detected with the standard 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

Tyramide stock solution (200X)

Add 100 µL DMSO to the vial of XFD488 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 cycles.

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 an appropriate concentration of secondary antibody-HRP working solution 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 NameEx/Em (nm)
17548Nuclear Blue™ DCS1350/461
17550Nuclear Green™ DCS1503/526
17551Nuclear Orange™ DCS1528/576
17552Nuclear Red™ DCS1642/660

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
XFD546 tyramide reagent *Same Structure to Alexa Fluor™ 546 tyramide*5615721120000.7910.210.12
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 19 citations: Citation Explorer
H3K9 lactylation in malignant cells facilitates CD8+ T cell dysfunction and poor immunotherapy response
Authors: Wang, Ruijie and Li, Chuwen and Cheng, Zhongyi and Li, Mingyu and Shi, Jianbo and Zhang, Zhiyuan and Jin, Shufang and Ma, Hailong
Journal: Cell Reports (2024)
The mammalian midbody and midbody remnant are assembly sites for RNA and localized translation
Authors: Park, Sungjin and Dahn, Randall and Kurt, Elif and Presle, Adrien and VanDenHeuvel, Kathryn and Moravec, Cara and Jambhekar, Ashwini and Olukoga, Olushola and Shepherd, Jason and Echard, Arnaud and others,
Journal: Developmental Cell (2023)
Stage-specific requirement for METTL3-dependent m6A modification during dental pulp stem cell differentiation
Authors: Luo, Haiyun and Liu, Wenjing and Zhou, Yachuan and Zhang, Yanli and Wu, Junrong and Wang, Ruolan and Shao, Longquan
Journal: Journal of Translational Medicine (2022): 1--15
The midbody and midbody remnant are assembly sites for RNA and active translation
Authors: Skop, Ahna and Park, Sungjin and Dahn, Randall and Kurt, Elif and Presle, Adrien and VandenHeuvel, Kathryn and Moravec, Cara and Jambhekar, Ashwini and Olukoga, Olushola and Shepherd, Jason and others,
Journal: (2022)
Cloning, pattern of gonadal soma-derived factor mRNA in the orange-spotted grouper, Epinephelus coioides
Authors: Huang, Jingjun and Wei, Qianhao and Zhao, Mi and Zhou, Libin and Shi, Herong and Zhang, Yong and Chen, Huapu
Journal: Aquaculture Reports (2021): 100754

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
Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis
Authors: Stack EC, Wang C, Roman KA, Hoyt CC.
Journal: Methods (2014): 46
Page updated on December 6, 2024

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Catalog Number11070
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Physical properties

Molecular weight

856.02

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.3

Correction Factor (280 nm)

0.11

Extinction coefficient (cm -1 M -1)

73000

Excitation (nm)

499

Emission (nm)

520

Quantum yield

0.921

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

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall, clear bottom