XFD546 tyramide reagent Same Structure to Alexa Fluor™ 546 tyramide

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

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-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

Overview SDS Protocol

Molecular weight

1282.88

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

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.

Platform

Fluorescence microscope

Excitation	Cy3/TRITC filter set
Emission	Cy3/TRITC filter set
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Cy3/TRITC filter set

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 546 tyramide stock solution (200X)

Add 100 µL of DMSO into the vial of XFD 546 tyramide conjugate to make 200X tyramide stock solution. Note: Make single use aliquots, and store unused 200X stock solution at 2-8 °C 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 546 tyramide working solution (1X)

Every 2 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 200 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. Note: Tris Buffer, pH=7.4 can be used. For optimal performance, use ReadiUse Tyramide (TSA)/Styramide (PSA) Optimized Reaction buffer (AAT Cat# 45090).

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

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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	77.95 µL	389.748 µL	779.496 µL	3.897 mL	7.795 mL
5 mM	15.59 µL	77.95 µL	155.899 µL	779.496 µL	1.559 mL
10 mM	7.795 µL	38.975 µL	77.95 µL	389.748 µL	779.496 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
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Spectrum

Open in Advanced Spectrum Viewer

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

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
XFD488 tyramide reagent Same Structure to Alexa Fluor™ 488 tyramide	499	520	73000	0.92¹	0.3	0.11
XFD594 tyramide reagent Same Structure to Alexa Fluor™ 594 tyramide	590	618	92000	0.66¹	0.43	0.56
XFD350 tyramide reagent Same Structure to Alexa Fluor™ 350 tyramide	343	441	19000	-	0.25	0.19
XFD568 tyramide reagent Same Structure to Alexa Fluor™ 568 tyramide	579	603	88000	0.69¹	0.45	0.46

Citations

View all 15 citations: Citation Explorer

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

Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters
Authors: Rees, J. S., Li, X. W., Perrett, S., Lilley, K. S., Jackson, A. P.
Journal: Curr Protoc Protein Sci (2017): 19 27 1-19 27 18

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification
Authors: J, undefined and ura, A., Hu, J., Wilk, R., Krause, H. M.
Journal: J Vis Exp (2017): se name="11070.enl" path="C:\Website\Referenc

Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System
Authors: Akama, K., Shirai, K., Suzuki, S.
Journal: Anal Chem (2016): 7123-9

Quantum dot-based FRET for sensitive determination of hydrogen peroxide and glucose using tyramide reaction
Authors: Huang, X., Wang, J., Liu, H., Lan, T., Ren, J.
Journal: Talanta (2013): 79-84

Gold nanoparticle-enzyme conjugates based FRET for highly sensitive determination of hydrogen peroxide, glucose and uric acid using tyramide reaction
Authors: Huang, X., Lan, T., Zhang, B., Ren, J.
Journal: Analyst (2012): 3659-66

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

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

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

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

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

Characterization of GABAergic neurons in the mouse lateral septum: a double fluorescence in situ hybridization and immunohistochemical study using tyramide signal amplification
Authors: Zhao C, Eisinger B, Gammie SC.
Journal: PLoS One (2013): e73750

Quantification of alpha-tubulin isotypes by sandwich ELISA with signal amplification through biotinyl-tyramide or immuno-PCR
Authors: Draberova E, Stegurova L, Sulimenko V, Hajkova Z, Draber P.
Journal: J Immunol Methods (2013): 63

Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: endogenous streptavidin-binding sites lead to false positive staining
Authors: Horling L, Neuhuber WL, Raab M.
Journal: J Neurosci Methods (2012): 124

Integrated tyramide and polymerization-assisted signal amplification for a highly-sensitive immunoassay
Authors: Yuan L, Xu L, Liu S.
Journal: Anal Chem (2012): 10737