XFD532 tyramide

Product key features

Ex/Em: 534/553 nm
Extinction coefficient: 81,000 cm^-1M^-1
Tyramide Signal Amplification (TSA): Facilitates ultrasensitive detection of low-abundance targets, ideal for IHC and ICC applications
Bright & Stable XFD532 Dye: Delivers high quantum yield with robust resistance to photobleaching and pH variations (4–10)

Product description

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. 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. XFD 532 tyramide contains the Alexa Fluor® 532 fluorophore that can be readily detected with the Alexa Fluor® 532/ATTO 532 filter set (Alexa Fluor® is the trademark of ThermoFisher). XFD 532 tyramide has intense yellow-orange fluorescence color.

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.

1. XFD 532 tyramide stock solution (100X)

Add 100 µL of DMSO into the vial of XFD 532 tyramide conjugate to make 100X tyramide stock solution.
Note Make single use aliquots, and store unused 100X stock solution at 2-8 ^oC in dark place.

2. H₂O₂ stock solution

Add 10 µL of 3% hydrogen peroxide (Not provided) to 90 µL of ddH₂O.
Note Prepare the 100X H₂O₂ solution fresh on the day of use.

PREPARATION OF WORKING SOLUTION

1. XFD 532 tyramide working solution (1X)

Every 1 mL of Reaction Buffer requires 10 µL of tyramide stock solution and 10 µL of H₂O₂ stock solution.
Note The tyramide provided is enough for 100 tests based on 100 µL of tyramide working solution needed per coverslip or per well in a 96-well microplate.
Note The tyramide working solution must be used within 2 hours after preparation and avoid direct exposure to light.

2. 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

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)
XFD532 acid Same Structure to Alexa Fluor™ 532 acid	534	553	81000	0.61¹	0.24	0.09
XFD514 tyramide	518	543	80000	-	0.31	0.18
XFD532 amine	534	553	81000	0.61¹	0.24	0.09
XFD532 azide	534	553	81000	0.61¹	0.24	0.09
XFD532 alkyne	534	553	81000	0.61¹	0.24	0.09
XFD532 TCO	534	553	81000	0.61¹	0.24	0.09
XFD532 Tetrazine	534	553	81000	0.61¹	0.24	0.09
Cy3 tyramide	555	569	150000¹	0.15¹	0.07	0.073
Cy5 tyramide	651	670	250000¹	0.27¹, 0.4²	0.02	0.03
Cy7 tyramide	756	779	250000	0.3	0.05	0.036
Fluorescein Tyramide	498	517	80000¹	0.7900¹, 0.95²	0.32	0.35
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References

View all 13 references: Citation Explorer

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

Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation.
Authors: Kuwajima, Masaaki and Ostrovskaya, Olga I and Cao, Guan and Weisberg, Seth A and Harris, Kristen M and Zemelman, Boris V
Journal: PloS one (2020): e0226797

NIR-labeled perfluoropolyether nanoemulsions for drug delivery and imaging.
Authors: O'Hanlon, Claire E and Amede, Konjit G and O'Hear, Meredith R and Janjic, Jelena M
Journal: Journal of fluorine chemistry (2012): 27-33

Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.
Authors: Clutter, Matthew R and Heffner, Garrett C and Krutzik, Peter O and Sachen, Kacey L and Nolan, Garry P
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2010): 1020-31

Methoxychlor and estradiol induce oxidative stress DNA damage in the mouse ovarian surface epithelium.
Authors: Symonds, Daniel A and Merchenthaler, Istvan and Flaws, Jodi A
Journal: Toxicological sciences : an official journal of the Society of Toxicology (2008): 182-7

Page updated on November 20, 2024

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Catalog Number	11072
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Physical properties

Molecular weight	847.06
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.24
Correction Factor (280 nm)	0.09
Extinction coefficient (cm ^-1 M ^-1)	81000
Excitation (nm)	534
Emission (nm)	553
Quantum yield	0.61¹

Storage, safety and handling

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