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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  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&nbsp;IgG and<strong><em>&nbsp;</em></strong>iFluor® 488 tyramide (Left) or Alexa Fluor&reg; 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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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
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iFluor® 350 goat anti-rabbit IgG (H+L)
iFluor® 405 goat anti-rabbit IgG (H+L)
iFluor® 488 goat anti-rabbit IgG (H+L)
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iFluor® 405 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 488 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 514 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 532 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 700 Styramide *Superior Replacement for Alexa Fluor 700 tyramide*
iFluor® 750 Styramide *Superior Replacement for Alexa Fluor 750 tyramide*
iFluor® 790 Styramide *Superior Replacement for Alexa Fluor 790 tyramide*
iFluor® 350 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 488 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 555 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 594 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 647 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
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iFluor® 810 goat anti-mouse IgG (H+L)
iFluor® 810 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 820 goat anti-mouse IgG (H+L)
iFluor® 820 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 840 goat anti-mouse IgG (H+L)
iFluor® 840 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 860 goat anti-mouse IgG (H+L)
iFluor® 860 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 800 goat anti-rabbit IgG (H+L)
iFluor® 800 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 810 goat anti-rabbit IgG (H+L)
iFluor® 810 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 820 goat anti-rabbit IgG (H+L)
iFluor® 820 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 840 goat anti-rabbit IgG (H+L)
iFluor® 840 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 860 goat anti-rabbit IgG (H+L)
iFluor® 860 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 840 maleimide
iFluor® 770 maleimide
iFluor® 780 maleimide
iFluor® 350 succinimidyl ester
iFluor® 405 succinimidyl ester
iFluor® 488 succinimidyl ester
iFluor® 514 succinimidyl ester
iFluor® 532 succinimidyl ester
iFluor® 555 succinimidyl ester
iFluor® 594 succinimidyl ester
iFluor® 633 succinimidyl ester
iFluor® 647 succinimidyl ester
iFluor® 660 succinimidyl ester
iFluor® 680 succinimidyl ester
iFluor® 700 succinimidyl ester
iFluor® 750 succinimidyl ester
iFluor® 610 succinimidyl ester
iFluor® 710 succinimidyl ester
iFluor® 790 succinimidyl ester
iFluor® 800 succinimidyl ester
iFluor® 810 succinimidyl ester
iFluor® 820 succinimidyl ester
iFluor® 860 succinimidyl ester
iFluor® 546 succinimidyl ester
iFluor® 568 succinimidyl ester
iFluor® 430 succinimidyl ester
iFluor® 450 succinimidyl ester
iFluor® 840 succinimidyl ester
iFluor® 560 succinimidyl ester
iFluor® 670 succinimidyl ester
iFluor® 460 succinimidyl ester
iFluor® 440 succinimidyl ester
iFluor® 665 succinimidyl ester
iFluor® 690 succinimidyl ester
iFluor® Ultra 594 succinimidyl ester
iFluor® Ultra 647 succinimidyl ester
iFluor® Ultra 750 succinimidyl ester
iFluor® 720 succinimidyl ester
iFluor® 740 succinimidyl ester
iFluor® 597 succinimidyl ester
iFluor® 770 succinimidyl ester
iFluor® 780 succinimidyl ester
iFluor® 570 succinimidyl ester
iFluor® 830 acid
iFluor® 830 maleimide
iFluor® 830 succinimidyl ester
iFluor® 405 azide
iFluor® 514 maleimide
iFluor® 660 maleimide
iFluor® 670 maleimide
iFluor® 675 succinimidyl ester
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iFluor® 750-Concanavalin A Conjugate
iFluor® 605 succinimidyl ester
iFluor® 605 maleimide
iFluor® 625 succinimidyl ester
iFluor® 625 maleimide
iFluor® 510 succinimidyl ester
iFluor® 510 maleimide
iFluor® 540 succinimidyl ester
iFluor® 540 maleimide
iFluor® 350-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 532-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 680-Wheat Germ Agglutinin (WGA) Conjugate
iFluor® 700-Wheat Germ Agglutinin (WGA) Conjugate
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iFluor® 350-dUTP *1 mM in Tris Buffer (pH 7.5)*
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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, 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® 488 tyramide contains the bright iFluor® 488 that can be readily detected with the standard FITC filter set. iFluor® dyes have higher florescence intensity, increased photostability, and enhanced water solubility, resulting in fluorescence signals with significantly higher precision and sensitivity. iFluor® 488 is an excellent replacement for Alexa Fluor® 488 tyramide (Alexa Fluor® is the trademark of ThermoFisher), FITC tyramide, or other comparable fluorescent tyramide conjugates.

Platform


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC 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

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
  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 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
  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 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.

  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.

    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.

  3. Use the appropriate filter set to visualize the signal from the Tyramide labeling.

Table 1. Products recommended for nucleus counterstain

Cat#Product NameEx/Em (nm)
17548Nuclear Blue™ DCS1350/461
17550Nuclear Green™ DCS1503/526
17551Nuclear Orange™ DCS1528/576
17552Nuclear Red™ DCS1642/660

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
/=x=

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

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 488 maleimide4915167500010.910.210.11
iFluor® 488 amine4915167500010.910.210.11
iFluor® 488 hydrazide4915167500010.910.210.11
iFluor® 488 azide4915167500010.910.210.11
iFluor® 488 alkyne4915167500010.910.210.11
iFluor® 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide and Opal 520*4915167500010.910.210.11
iFluor® 555 Tyramide55757010000010.6410.230.14
iFluor® 647 Tyramide65667025000010.2510.030.03
iFluor® 350 Tyramide3454502000010.9510.830.23
iFluor® 546 Tyramide54155710000010.6710.250.15
iFluor® 568 Tyramide56858710000010.5710.340.15
iFluor® 594 Tyramide58760320000010.5310.050.04
iFluor® 488 TCO4915167500010.910.210.11
iFluor® 488 Tetrazine4915167500010.910.210.11
iFluor®488-dUTP *1 mM in TE Buffer (pH 7.5)*4915167500010.910.210.11
iFluor® 633 tyramide64065425000010.2910.0620.044
iFluor® 430 Tyramide *Superior Replacement for Opal 480*4334984000010.7810.680.3
iFluor® 450 Tyramide *Superior Replacement for Opal 480*4515024000010.8210.450.27
iFluor® 680 Tyramide *Superior Replacement for Opal 690*68470122000010.2310.0970.094
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Images


Citations


View all 18 citations: Citation Explorer
PIM1 is a potential therapeutic target for the leukemogenic effects mediated by JAK/STAT pathway mutations in T-ALL/LBL
Authors: Lahera, Antonio and Vela-Mart{\'\i}n, Laura and Fern{\'a}ndez-Navarro, Pablo and Llamas, Pilar and L{\'o}pez-Lorenzo, Jos{\'e} L and Cornago, Javier and Santos, Javier and Fern{\'a}ndez-Piqueras, Jos{\'e} and Villa-Morales, Mar{\'\i}a
Journal: npj Precision Oncology (2024): 152
Contribution of Tregs to the promotion of constructive remodeling after decellularized extracellular matrix material implantation
Authors: Jiang, Hongjing and Sun, Xuheng and Wu, Yindi and Xu, Jianyi and Xiao, Cong and Liu, Qing and Fang, Lijun and Liang, Yuanfeng and Zhou, Jiahui and Wu, Yueheng and others,
Journal: Materials Today Bio (2024): 101151
IKIP downregulates THBS1/FAK signaling to suppress migration and invasion by glioblastoma cells
Authors: Zhu, Zhaoying and Hu, Yanjia and Ye, Feng and Teng, Haibo and You, Guoliang and Zeng, Yunhui and Tian, Meng and Xu, Jianguo and Li, Jin and Liu, Zhiyong and others,
Journal: Oncology Research (2024): 1173
Cell growth and mitochondrial anomalies in induced pluripotent stem cells with Presenilin 1 mutation
Authors: Hamam, Rimi and Hanna, Roy and Barabino, Andrea and Serhani, Dounya and Tavares, Erika and {\'E}lise, H{\'e}on and Bernier, Gilbert
Journal: (2024)
Cross-species single-cell analysis uncovers the immunopathological mechanisms associated with IgA nephropathy progression
Authors: Chen, Xizhao and Wang, Tiantian and Chen, Lei and Zhao, Yinghua and Deng, Yiyao and Shen, Wanjun and Li, Lin and Yin, Zhong and Zhang, Chaoran and Cai, Guangyan and others,
Journal: JCI Insight (2024)
PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells
Authors: Lacher, Sebastian B and D{\"o}rr, Janina and de Almeida, Gustavo P and H{\"o}nninger, Julian and Bayerl, Felix and Hirschberger, Anna and Pedde, Anna-Marie and Meiser, Philippa and Ramsauer, Lukas and Rudolph, Thomas J and others,
Journal: Nature (2024): 1--9
Poor clinical outcomes and immunoevasive contexture in CD161+ CD8+ T cells barren human pancreatic cancer
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Journal: Journal for Immunotherapy of Cancer (2024)
DHCR24 insufficiency promotes vascular endothelial cell senescence and endothelial dysfunction via inhibition of Caveolin-1/ERK signaling
Authors: Li, Han and Yang, Zhen and Liang, Wukaiyang and Nie, Hao and Guan, Yuqi and Yang, Ni and Ji, Tianyi and Liu, Yu and Huang, Yi and Zhang, Le and others,
Journal: The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences (2024): glae059
Intratumoral injection of interferon gamma promotes the efficacy anti-PD1 treatment in colorectal cancer
Authors: Tang, Yang and Wei, Jingsun and Ge, Xiaoxu and Yu, Chengxuan and Lu, Wei and Qian, Yucheng and Yang, Hang and Fu, Dongliang and Fang, Yimin and Zhou, Yinyi and others,
Journal: Cancer Letters (2024): 216798
Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis
Authors: Hu, Xingxing and Zou, Menglin and Zheng, Weishuai and Zhu, Minghui and Hou, Qinhui and Gao, Han and Zhang, Xin and Liu, Yuan and Cheng, Zhenshun
Journal: Respiratory Research (2024): 1--13

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