XFD532 tyramide

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

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

Overview SDS Protocol

Platform

Fluorescence microscope

Excitation	Cy3/TRITC filter set
Emission	Cy3/TRITC filter set
Recommended plate	Black wall/clear bottom

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

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of XFD532 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	118.055 µL	590.277 µL	1.181 mL	5.903 mL	11.806 mL
5 mM	23.611 µL	118.055 µL	236.111 µL	1.181 mL	2.361 mL
10 mM	11.806 µL	59.028 µL	118.055 µL	590.277 µL	1.181 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

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

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
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

Images

Figure 1. Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA ™ amplified methods. Human lung adenocarcinoma positive tissue sections were stained with Mouse anti-EpCAM or Control Mouse IgG antibody and then incubated with polyHRP-labeled Goat anti-Mouse IgG secondary antibody followed by XFD 532 tyramide (Cat#11072).

Figure 2. Chemical structure for XFD 532 tyramide.

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

Genotyping of phenotypically defined cells in neoplasia: enhanced immunoFISH via tyramide signal amplification (TSA) segregates immunophenotypically-defined cell populations for gated genotyping.
Authors: Tubbs, Raymond R and Das, Kingshuk and Cook, James R and Pettay, James D and Roche, Patrick C and Grogan, Thomas
Journal: Journal of molecular histology (2007): 129-34

A CARD-FISH protocol for the identification and enumeration of epiphytic bacteria on marine algae.
Authors: Tujula, Niina A and Holmström, Carola and Mussmann, Marc and Amann, Rudolf and Kjelleberg, Staffan and Crocetti, Gregory R
Journal: Journal of microbiological methods (2006): 604-7

Novel oxidative self-anchoring fluorescent substrates for the histochemical localization of endogenous and immunobound peroxidase activity.
Authors: Krieg, Reimar and Halbhuber, Karl-Jürgen
Journal: Journal of molecular histology (2004): 471-87

Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.
Authors: Warsen, Adelaide E and Krug, Melissa J and LaFrentz, Stacey and Stanek, Danielle R and Loge, Frank J and Call, Douglas R
Journal: Applied and environmental microbiology (2004): 4216-21

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.
Authors: Panicker, Gitika and Call, Douglas R and Krug, Melissa J and Bej, Asim K
Journal: Applied and environmental microbiology (2004): 7436-44