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iFluor® 488 tyramide

Immunofluorescent image of paraffin-embedded human lung carcinoma labeled with  EpCAM Rabbit mAb followed with HRP-labeled goat anti-rabbit IgG (H+L) (Cat#16793) . The signal was developed with iFluor® 488 tyramide or Alexa Fluor™ 488 Tyramide Reagent (Green). Cells were also counterstained with DAPI (Blue).
Immunofluorescent image of paraffin-embedded human lung carcinoma labeled with  EpCAM Rabbit mAb followed with HRP-labeled goat anti-rabbit IgG (H+L) (Cat#16793) . The signal was developed with iFluor® 488 tyramide or Alexa Fluor™ 488 Tyramide Reagent (Green). Cells were also counterstained with DAPI (Blue).
Immunofluorescent image of paraffin-embedded human lung carcinoma labeled with Pan-Keratin Mouse mAb followed with HRP-labeled goat anti-mouse IgG (H+L) (Cat#16728). The signal was developed with iFluor® 488 tyramide (Green). Cells were also counterstained with DAPI (Blue).
<strong>Superior sensitivity with iFluor® 488 tyramide.</strong> HeLa cells were incubated with primary anti-tubulin antibodies followed by detection with HRP-Goat anti-Mouse IgG and<strong><em> </em></strong>iFluor® 488 tyramide (Left) or Alexa Fluor® 488 tyramide (Right). Fluorescence images were taken on a Keyence BZ-X710 fluorescence microscope equipped with a FITC filter set.
Formalin-fixed, paraffin-embedded (FFPE) human lung adenocarcinoma tissue was incubated with an anti-EpCAM primary antibody, and an HRP conjugated anti-mouse secondary antibody. TSA signal was developed by incubation of tissue section with 5 µg/mL of iFluor® 488 tyramide (Cat No. 45100) for 10 minutes. Images were acquired on a confocal microscope equipped with a GFP filter set.
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Catalog Number45100
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
Molecular weight531.47
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.21
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)491
Emission (nm)516
Quantum yield0.91
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
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
531.47
Correction Factor (260 nm)
0.21
Correction Factor (280 nm)
0.11
Extinction coefficient (cm -1 M -1)
750001
Excitation (nm)
491
Emission (nm)
516
Quantum yield
0.91
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. iFluor® 488 tyramide contains the extremely bright and photostable iFluor® 488 that can be readily detected with the standard FITC filter set.

Platform


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)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 vial and mix well. Note: Unused Tyramide stock solution can be stored at 2-8 °C.

PREPARATION OF WORKING SOLUTION

Tyramide working solution (1X)
Add 100 µL of Tyramide stock solution into 20 mL of buffer of your choice containing 0.003% H2O2. Note: Tris Buffer, pH=7.4 can be used for similar performance. Note: Tyramide working solution should be used immediately and made fresh on the day of use. Note: 20 mL solution is good for 200 tests.

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-tissueimmunohistochemistry-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

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 488 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 mg0.5 mg1 mg5 mg10 mg
1 mM188.157 µL940.787 µL1.882 mL9.408 mL18.816 mL
5 mM37.631 µL188.157 µL376.315 µL1.882 mL3.763 mL
10 mM18.816 µL94.079 µL188.157 µL940.787 µL1.882 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.21
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)491
Emission (nm)516
Quantum yield0.91

Citations


View all 4 citations: Citation Explorer
NEIL3-deficiency increases gut permeability and contributes to a pro-atherogenic metabolic phenotype
Authors: Karlsen, Tom Rune and Kong, Xiang Yi and Holm, Sverre and Quiles-Jim{\'e}nez, Ana and Dahl, Tuva B and Yang, Kuan and Sagen, Ellen L and Skarpengland, Tonje and S {\O}gaard, Jonas D and Holm, Kristian and others,
Journal: Scientific Reports (2021): 1--10
CD95/Fas protects triple negative breast cancer from anti-tumor activity of NK cells
Authors: Qadir, Abdul S and Gu{\'e}gan, Jean Philippe and Ginestier, Christophe and Chaibi, Assia and Bessede, Alban and Charafe-Jauffret, Emmanuelle and Macario, Manon and Lavou{\'e}, Vincent and de la Motte Rouge, Thibault and Law, Calvin and others,
Journal: Iscience (2021): 103348
Accelerated onset of cnS prion disease in mice co-infected with a gastrointestinal helminth pathogen during the preclinical phase
Authors: Donaldson, David S and Bradford, Barry M and Else, Kathryn J and Mabbott, Neil A
Journal: Scientific reports (2020): 1--17
Discrimination of prion strain targeting in the central nervous system via reactive astrocyte heterogeneity in CD44 expression
Authors: Bradford, Barry M and Wijaya, Christianus AW and Mabbott, Neil A
Journal: Frontiers in cellular neuroscience (2019): 411

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