iFluor® 514 Styramide *Superior Replacement for Opal 540*
Ordering information
Price | |
Catalog Number | |
Unit Size | |
Quantity |
Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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 | 1195.24 |
Solvent | DMSO |
Spectral properties
Absorbance (nm) | 511 |
Correction Factor (260 nm) | 0.265 |
Correction Factor (280 nm) | 0.116 |
Extinction coefficient (cm -1 M -1) | 750001 |
Excitation (nm) | 511 |
Emission (nm) | 527 |
Quantum yield | 0.831 |
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 | SDSProtocol |
Molecular weight 1195.24 | Absorbance (nm) 511 | Correction Factor (260 nm) 0.265 | Correction Factor (280 nm) 0.116 | Extinction coefficient (cm -1 M -1) 750001 | Excitation (nm) 511 | Emission (nm) 527 | Quantum yield 0.831 |
Power Styramide™ Signal Amplification (PSA™) system is one of the most sensitive methods that can detect extremely low-abundance targets in cells and tissues with improved fluorescence signal 10-50 times higher than the widely used tyramide (TSA) reagents. In combination with our superior iFluor® dyes that have higher fluorescence intensity, increased photostability, and enhanced water solubility, the iFluor® dye-labeled Styramide™ conjugates can generate fluorescence signal with significantly higher precision and sensitivity (more than 100 times) than standard ICC/IF/IHC. PSA utilizes the catalytic activity of horseradish peroxidase (HRP) for the covalent deposition of fluorophores in situ. PSA radicals have much higher reactivity than tyramide radicals, making the PSA system much faster, more robust, and more sensitive than traditional TSA reagents. Compared to tyramide reagents, the Styramide™ conjugates can label the target at higher efficiency and thus generate a significantly higher fluorescence signal. Styramide™ conjugates also allow substantially less consumption of primary antibodies compared to the standard directly conjugate method or tyramide amplification with the same level of sensitivity. iFluor® 514 Styramide is optimized to be a superior replacement for Opal Polaris 540 or other spectrally similar fluorescent tyramide conjugates or TSA reagents.
Platform
Fluorescence microscope
Excitation | FITC filter set |
Emission | FITC 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 Styramide™ 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. iFluor™ 514 Styramide stock solution (100X)
Add 100 µL of DMSO into the vial of iFluor™ 514 Styramide conjugate to make 100X Styramide stock solution. Note: Make single use aliquots, and store unused 100X stock solution at 2-8 °C 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. iFluor™ 514 Styramide working solution (1X)
Every 1 mL of Reaction Buffer requires 10 µL of Styramide stock solution and 10 µL of H2O2 stock solution. Note: The Styramide provided is enough for 100 tests based on 100 µL of Styramide working solution needed per coverslip or per well in a 96-well microplate. Note: The Styramide 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.
Protocol can be found at
https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html
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 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
- 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.
Styramide labeling
- Prepare and apply 100 µL of Styramide 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 Styramide. You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use lower concentration of Styramide 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.
- Use the appropriate filter set to visualize the signal from the Styramide labeling.
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® 514 Styramide *Superior Replacement for Opal 540* 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 | 83.665 µL | 418.326 µL | 836.652 µL | 4.183 mL | 8.367 mL |
5 mM | 16.733 µL | 83.665 µL | 167.33 µL | 836.652 µL | 1.673 mL |
10 mM | 8.367 µL | 41.833 µL | 83.665 µL | 418.326 µL | 836.652 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
Absorbance (nm) | 511 |
Correction Factor (260 nm) | 0.265 |
Correction Factor (280 nm) | 0.116 |
Extinction coefficient (cm -1 M -1) | 750001 |
Excitation (nm) | 511 |
Emission (nm) | 527 |
Quantum yield | 0.831 |
Product Family
Images
Figure 1. Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA™ 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 iFluor® 514 Styramide™ (Cat. 45022).
References
View all 50 references: Citation Explorer
A novel chemiluminescence imaging immunosensor for prostate specific antigen detection based on a multiple signal amplification strategy.
Authors: Zhao, Li-Zhen and Fu, Yi-Zhuo and Ren, Shu-Wei and Cao, Jun-Tao and Liu, Yan-Ming
Journal: Biosensors & bioelectronics (2021): 112729
Authors: Zhao, Li-Zhen and Fu, Yi-Zhuo and Ren, Shu-Wei and Cao, Jun-Tao and Liu, Yan-Ming
Journal: Biosensors & bioelectronics (2021): 112729
Imaging sensor array coupled with dual-signal amplification strategy for ultrasensitive chemiluminescence immunoassay of multiple mycotoxins.
Authors: Zong, Chen and Jiang, Fan and Wang, Xiaoyu and Li, Ping and Xu, Linru and Yang, Hua
Journal: Biosensors & bioelectronics (2021): 112998
Authors: Zong, Chen and Jiang, Fan and Wang, Xiaoyu and Li, Ping and Xu, Linru and Yang, Hua
Journal: Biosensors & bioelectronics (2021): 112998
Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures.
Authors: Schafer, Johanna M and Pietenpol, Jennifer A
Journal: Bio-protocol (2020): e3677
Authors: Schafer, Johanna M and Pietenpol, Jennifer A
Journal: Bio-protocol (2020): e3677
Cascade signal amplification for sensitive detection of exosomes by integrating tyramide and surface-initiated enzymatic polymerization.
Authors: Huang, Zhipeng and Lin, Qiuyuan and Yang, Bin and Ye, Xin and Chen, Hui and Weng, Wenhao and Kong, Jilie
Journal: Chemical communications (Cambridge, England) (2020): 12793-12796
Authors: Huang, Zhipeng and Lin, Qiuyuan and Yang, Bin and Ye, Xin and Chen, Hui and Weng, Wenhao and Kong, Jilie
Journal: Chemical communications (Cambridge, England) (2020): 12793-12796
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
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)
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
Authors: Lauter, Gilbert and Söll, Iris and Hauptmann, Giselbert
Journal: Methods in molecular biology (Clifton, N.J.) (2020): 397-409
Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins.
Authors: Dopie, Joseph and Sweredoski, Michael J and Moradian, Annie and Belmont, Andrew S
Journal: The Journal of cell biology (2020)
Authors: Dopie, Joseph and Sweredoski, Michael J and Moradian, Annie and Belmont, Andrew S
Journal: The Journal of cell biology (2020)
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)
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)
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
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
Application notes
A New Protein Crosslinking Method for Labeling and Modifying Antibodies
A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Biotin Labeling Molecules and Their Biological Applications
Buccutite™ Bioconjugation Technology
A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
Biotin Labeling Molecules and Their Biological Applications
Buccutite™ Bioconjugation Technology
FAQ
How can I lyse my cells without lysing the nuclear membrane?
What are the differences between calcium ion indicators: Cal 520, Cal 520FF, and Cal 520N?
How do I make an AM ester stock solution?
Can we fix cells with glutaraldehyde and then stain with fluorescent phalloidin?
What is the difference between FluoroQuest Anti-fading Kit I and FluoroQuest Anti-fading Kit II?
What are the differences between calcium ion indicators: Cal 520, Cal 520FF, and Cal 520N?
How do I make an AM ester stock solution?
Can we fix cells with glutaraldehyde and then stain with fluorescent phalloidin?
What is the difference between FluoroQuest Anti-fading Kit I and FluoroQuest Anti-fading Kit II?