iFluor® 568 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	1195.55
Solvent	DMSO

Spectral properties

Correction Factor (260 nm)	0.34
Correction Factor (280 nm)	0.15
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	568
Emission (nm)	587
Quantum yield	0.57¹

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

1195.55

Correction Factor (260 nm)

0.34

Correction Factor (280 nm)

0.15

Extinction coefficient (cm ^-1 M ^-1)

100000¹

Excitation (nm)

568

Emission (nm)

587

Quantum yield

0.57¹

For many immunohistochemical (IHC) applications, traditional enzymatic amplification procedures are sufficient for achieving adequate antigen detection. However, several factors limit their sensitivity and utility. 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 results in the ability to detect low-abundance targets with ultrasensitive precision and reduces the amount of antibodies and hybridization probes needed. In IHC applications, this method can also enhance sensitivity in cases where the primary antibody dilution needs to be increased to reduce nonspecific background signals or overcome weak immunolabeling due to suboptimal fixation procedures or low levels of target expression. The iFluor® 568 tyramide contains the bright iFluor® 568 that can be readily detected with the standard TRITC or Cy3 filter set. iFluor® dyes have higher fluorescence intensity, increased photostability, and enhanced water solubility, resulting in fluorescence signals with significantly higher precision and sensitivity. iFluor® 568 is an excellent replacement for Alexa Fluor® 568 tyramide (Alexa Fluor® is the trademark of ThermoFisher) or other comparable fluorescent tyramide conjugates.

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

Tyramide stock solution (200X)

Add 100 µL of DMSO to the vial of iFluor® tyramide and mix well.

Note: Make single-use aliquots and store unused 200X stock solution at 2-8 °C, protected from light. Avoid repeat freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

Tyramide working solution (1X)

Add 100 µL of the tyramide stock solution into 20 mL of a buffer of your choice containing 0.003% H₂O₂.

Note: For optimal performance, use Tris Buffer, pH=7.4.

Note: A 20 mL solution is good for 200 tests. The tyramide working solution should be used immediately and made fresh on the day of use. Avoid direct exposure to light.

Secondary antibody-HRP working solution

Make an appropriate concentration of secondary antibody-HRP working solution 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 the preferred specific solution/protocol as needed. A 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 a non-specific signal, you can shorten the incubation time with the tyramide reagent. You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use a lower concentration of the tyramide reagent 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.

Note: To ensure optimal results, it is recommended to use either ReadiUse™ microscope mounting solution (Cat. 20009) or FluoroQuest™ TSA/PSA Antifade Mounting Medium *Optimized for Tyramide and Styramide Imaging* (Cat. 44890) instead of Vectashield® mounting media. There are instances where Vectashield® mounting media may not be suitable for certain TSA/PSA conjugates.
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® 568 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	83.644 µL	418.218 µL	836.435 µL	4.182 mL	8.364 mL
5 mM	16.729 µL	83.644 µL	167.287 µL	836.435 µL	1.673 mL
10 mM	8.364 µL	41.822 µL	83.644 µL	418.218 µL	836.435 µL

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)	0.34
Correction Factor (280 nm)	0.15
Extinction coefficient (cm ^-1 M ^-1)	100000¹
Excitation (nm)	568
Emission (nm)	587
Quantum yield	0.57¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 488 tyramide	491	516	75000¹	0.9¹	0.21	0.11
iFluor® 568 maleimide	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 568 Styramide Superior Replacement for Alexa Fluor 568 tyramide	568	587	100000¹	0.57¹	0.34	0.15
iFluor® 555 Tyramide	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 647 Tyramide	656	670	250000¹	0.25¹	0.03	0.03
iFluor® 350 Tyramide	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 546 Tyramide	541	557	100000¹	0.67¹	0.25	0.15
iFluor® 594 Tyramide	587	603	200000¹	0.53¹	0.05	0.04
iFluor® 633 tyramide	640	654	250000¹	0.29¹	0.062	0.044
iFluor® 430 Tyramide Superior Replacement for Opal 480	433	498	40000¹	0.78¹	0.68	0.3
iFluor® 450 Tyramide Superior Replacement for Opal 480	451	502	40000¹	0.82¹	0.45	0.27
iFluor® 680 Tyramide Superior Replacement for Opal 690	684	701	220000¹	0.23¹	0.097	0.094
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Images

Figure 1. Microtubules of fixed HeLa cells were labeled with anti-α tubulin mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using Alexa Fluor® 568 tyramide or iFluor® 568 tyramide (Cat No. 45106) and detected with a TRITC/Cy3 filter set. iFluor® 568 tyramide shows significantly higher fluorescence intensity than Alexa Fluor® 568 tyramide under the same conditions.

Figure 2. Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using iFluor® 568 tyramide (Cat No. 45106) or Alexa Fluor® 568 tyramide and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).

Figure 3. Chemical structure for iFluor® 568 Tyramide.

References

View all 50 references: Citation Explorer

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals - Using Unconjugated Primary Antibodies from the Same Host Species.
Authors: Lyu, Qiongxia and Zheng, Huifei Sophia and Laprocina, Karly and Huang, Chen-Che Jeff
Journal: Journal of visualized experiments : JoVE (2020)

Detection of Cytokine Receptors Using Tyramide Signal Amplification for Immunofluorescence.
Authors: Wang, Herui and Pangilinan, Ryan L and Zhu, Yan
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 89-97

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies.
Authors: Parra, Edwin Roger and Jiang, Mei and Solis, Luisa and Mino, Barbara and Laberiano, Caddie and Hernandez, Sharia and Gite, Swati and Verma, Anuj and Tetzlaff, Michael and Haymaker, Cara and Tamegnon, Auriole and Rodriguez-Canales, Jaime and Hoyd, Clifford and Bernachez, Chantale and Wistuba, Ignacio
Journal: Cancers (2020)

Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced Tyramide Signal Amplification and Its Combination with Immunofluorescent Protein Visualization in Zebrafish.
Authors: Lauter, Gilbert and Söll, Iris and Hauptmann, Giselbert
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 397-409

Optimization of prostate cancer cell detection using multiplex tyramide signal amplification.
Authors: Roy, Sounak and Axelrod, Haley D and Valkenburg, Kenneth C and Amend, Sarah and Pienta, Kenneth J
Journal: Journal of cellular biochemistry (2019): 4804-4812

Highly Sensitive Detection of PCV2 Based on Tyramide Signals and GNPL Amplification.
Authors: Zhang, Shouping and Hu, Bin and Xia, Xiaojing and Xu, Yanzhao and Hang, Bolin and Jiang, Jinqing and Hu, Jianhe
Journal: Molecules (Basel, Switzerland) (2019)

DNAzyme Catalyzed Tyramide Depositing Reaction for In Situ Imaging of Protein Status on the Cell Surface.
Authors: Xu, Lulu and Liu, Shengchun and Yang, Tiantian and Shen, Yifan and Zhang, Yuhong and Huang, Lizhen and Zhang, Lutan and Ding, Shijia and Song, Fangzhou and Cheng, Wei
Journal: Theranostics (2019): 1993-2002

Cancer immunophenotyping by seven-colour multispectral imaging without tyramide signal amplification.
Authors: Ijsselsteijn, Marieke E and Brouwer, Thomas P and Abdulrahman, Ziena and Reidy, Eileen and Ramalheiro, Ana and Heeren, A Marijne and Vahrmeijer, Alexander and Jordanova, Ekaterina S and de Miranda, Noel Fcc
Journal: The journal of pathology. Clinical research (2019): 3-11

Comparative Tyramide-FISH mapping of the genes controlling flavor and bulb color in Allium species revealed an altered gene order.
Authors: Khrustaleva, Ludmila and Kudryavtseva, Natalia and Romanov, Dmitry and Ermolaev, Aleksey and Kirov, Ilya
Journal: Scientific reports (2019): 12007

An ultrasensitive electrochemical immunosensor for procalcitonin detection based on the gold nanoparticles-enhanced tyramide signal amplification strategy.
Authors: Liu, Pei and Li, Chao and Zhang, Ruixuan and Tang, Qing and Wei, Jia and Lu, Yan and Shen, Pingping
Journal: Biosensors & bioelectronics (2019): 543-550