iFluor™ 488 tyramide

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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).
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Overview

Ex/Em (nm)491/516
MW531.47
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Horseradish Peroxidase (HRP)
Related
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.




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.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Wavelength (nm)





Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
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

Cat. #

Product Name

Unit

Ex (nm)

Em (nm)

11070

AF 488 Tyramide reagent

200 slides

491

518

11075

AF 546 Tyramide reagent

200 slides

554

570

11082

AF 594 Tyramide reagent

200 slides

590

617

11061

Azido-Cy5 Tyramide

1 mg

644

665

11065

Cy3 Tyramide

1 mg

555

565

11066

Cy5 Tyramide

1 mg

644

665

45100

iFluorTM 488 Tyramide

200 slides

491

514

45105

iFluorTM 555 Tyramide

200 slides

552

567

45110

iFluorTM 647 Tyramide

200 slides

649

665

Key parameters
Instrument:Fluorescence microscope
Excitation:FITC filter set
Emission:FITC filter set
Recommended plate:Black wall/clear bottom
Instrument specification(s):FITC filter set
Preparation of stock solution
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 2-8o 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% H2O2Note: Tris Buffer, pH=7.4 can be used for optimal 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-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

Example data analysis and figures

Figure 1. 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).
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.









References

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

Tyramide Signal Amplification for Immunofluorescent Enhancement
Authors: Faget L, Hnasko TS.
Journal: Methods Mol Biol (2015): 161

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

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

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

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

Development of a near-infrared fluorescence ELISA method using tyramide signal amplification
Authors: Gong H, Cradduck M, Cheung L, Olive DM.
Journal: Anal Biochem (2012): 27

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


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