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XFD532 tyramide

Chemical structure for XFD 532 tyramide.
Chemical structure for XFD 532 tyramide.
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA<strong> ™</strong> 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).
Ordering information
Price ()
Catalog Number11072
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
SolventDMSO
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 yield0.611
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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Show More (51)

OverviewpdfSDSpdfProtocol


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
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.

Platform


Fluorescence microscope

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

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.

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
  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 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
  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 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.
  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.
  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

Spectrum


Open in Advanced Spectrum Viewer
spectrum

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 yield0.611

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
XFD532 acid *Same Structure to Alexa Fluor™ 532 acid*534553810000.611--
XFD514 tyramide51854380000-0.310.18
Cy3 tyramide55556915000010.1510.070.073
Cy5 tyramide65167025000010.271, 0.420.020.03
Biotinyl tyramide------
Cy7 tyramide7567792500000.30.050.036
Fluorescein Tyramide4985178000010.79001, 0.9520.320.275

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