ReadiCleave™ iFluor® 647 Styramide
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Catalog Number | |
Unit Size | |
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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 |
Molecular weight | 1580.98 |
Solvent | DMSO |
Correction Factor (260 nm) | 0.03 |
Correction Factor (280 nm) | 0.03 |
Correction Factor (656 nm) | 0.0793 |
Extinction coefficient (cm -1 M -1) | 2500001 |
Excitation (nm) | 656 |
Emission (nm) | 670 |
Quantum yield | 0.251 |
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 |
Overview | ![]() ![]() |
Molecular weight 1580.98 | Correction Factor (260 nm) 0.03 | Correction Factor (280 nm) 0.03 | Correction Factor (656 nm) 0.0793 | Extinction coefficient (cm -1 M -1) 2500001 | Excitation (nm) 656 | Emission (nm) 670 | Quantum yield 0.251 |
Platform
Fluorescence microscope
Excitation | FITC filter set |
Emission | FITC filter set |
Recommended plate | Black wall/clear bottom |
Example protocol
AT A GLANCE
Fix/permeabilize/block cells or tissue.
Add the primary antibody in blocking buffer.
Add the HRP-conjugated secondary antibody.
Prepare the Styramide™ working solution and apply it to cells or tissue. Incubate at room temperature for 5-10 minutes.
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
To prepare a 100X stock solution of ReadiCleave™ iFluor® 647 Styramide, add 100 µL of DMSO to the vial containing the conjugate.
Note: Prepare single-use aliquots of the 100X stock solution and store any unused portions at 2-8°C, protected from light. Avoid freeze-thaw cycles.
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
For every 1 mL of Reaction Buffer, add 10 µL of ReadiCleave™ iFluor® 647 Styramide stock solution and 10 µL of H2O2 stock solution.
Note: The provided ReadiCleave™ iFluor® Styramide is sufficient for 100 tests, with each test requiring 100 µL of ReadiCleave™ iFluor® Styramide working solution per coverslip or per well in a 96-well microplate.
Note: The ReadiCleave™ iFluor® Styramide working solution must be used within 2 hours after preparation and avoid direct exposure to light.
Prepare the secondary antibody-HRP working solution according to the manufacturer's instructions.
SAMPLE EXPERIMENTAL PROTOCOL
This protocol is applicable for both cells and tissues staining.
Fix the cells or tissue using 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.
Deparaffinize and dehydrate the tissue following standard IHC protocols. Then, perform antigen retrieval using the preferred specific solution and protocol. Detailed instructions for the protocol can be found at:
https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html
Optional: Quench endogenous peroxidase activity by incubating the cell or tissue sample in a peroxidase quenching solution (e.g., 3% hydrogen peroxide) for 10 minutes. Rinse the sample twice with PBS at room temperature.
Optional: If using HRP-conjugated streptavidin, it is recommended to block endogenous biotin with a biotin blocking buffer.
Block the sample using your preferred blocking solution, such as PBS with 1% BSA, for 30 minutes at 4°C.
Remove the blocking solution. Add the primary antibody, diluted in the recommended antibody diluent, and incubate for 60 minutes at room temperature or overnight at 4 °C.
Wash with PBS three times for 5 minutes each.
Apply 100 µL of the secondary antibody-HRP working solution to each sample and incubate at room temperature for 60 minutes.
Note: Incubation time and concentration can be varied depending on the signal intensity.
Wash with PBS three times for 5 minutes each.
Prepare 100 µL of ReadiCleave™ iFluor® 647 Styramide working solution and apply it to each sample. Allow the samples to incubate at room temperature for 5-10 minutes.
Note: If you observe a non-specific signal, you can shorten the incubation time with Styramide. It is important to optimize the incubation period using positive and negative control samples at various time points. Additionally, you can use a lower concentration of Styramide in the working solution.
Rinse with PBS three times.
Prepare a 1X working solution, add 200 μL of ReadiCleave™ AML Cleavage Buffer (Cat. 7510, not provided) into 800 μL of ddH2O, and mix thoroughly.
Note: For optimal results, use this solution within a few hours of preparation.
Add 100 µL of ReadiCleave™ AML Cleavage Buffer working solution to the tissue or cell samples.
Note: Add a sufficient amount of ReadiCleave™ AML Cleavage Buffer working solution to ensure that the samples are fully submerged.
Heat the samples at 60°C for 60 minutes.
Remove the ReadiCleave™ AML Cleavage Buffer working solution and briefly rinse the samples with PBST.
Reprocess the tissue samples beginning with the Antigen Retrieval step in your IHC staining protocol.
For optimal results, counterstain the cell or tissue samples as needed. AAT offers a range of nucleus counterstain reagents, which are detailed in Table 1. Please follow the instructions provided with each reagent.
Mount the coverslip using an anti-fade mounting medium to prevent fading.
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. Vectashield® mounting media may not be compatible with some TSA/PSA conjugates.
Use the appropriate filter set to visualize the signal from the counterstain.
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
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 63.252 µL | 316.26 µL | 632.519 µL | 3.163 mL | 6.325 mL |
5 mM | 12.65 µL | 63.252 µL | 126.504 µL | 632.519 µL | 1.265 mL |
10 mM | 6.325 µL | 31.626 µL | 63.252 µL | 316.26 µL | 632.519 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
![spectrum](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fspectra%2Fifluor_647.png&w=2048&q=50)
Spectral properties
Correction Factor (260 nm) | 0.03 |
Correction Factor (280 nm) | 0.03 |
Correction Factor (656 nm) | 0.0793 |
Extinction coefficient (cm -1 M -1) | 2500001 |
Excitation (nm) | 656 |
Emission (nm) | 670 |
Quantum yield | 0.251 |
Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) |
ReadiCleave™ iFluor® 488 Styramide | 491 | 516 | 750001 | 0.91 | 0.21 | 0.11 |
ReadiCleave™ iFluor® 546 Styramide | 541 | 557 | 1000001 | 0.671 | 0.25 | 0.15 |
References
Authors: Liu, Wen-Jing and Wang, Lu-Yao and Sheng, Zhimei and Zhang, Baogang and Zou, Xiaoran and Zhang, Chun-Yang
Journal: Biosensors & bioelectronics (2023): 115645
Authors: Huang, Xiangyi and Wang, Jinjie and Liu, Heng and Lan, Tao and Ren, Jicun
Journal: Talanta (2013): 79-84
Authors: Nakamura, Ayako and Uchihara, Toshiki
Journal: Journal of neuroscience methods (2004): 67-70
Authors: Pougnard, Claire and Catala, Philippe and Drocourt, Jean-Louis and Legastelois, Stephane and Pernin, Pierre and Pringuez, Emmanuelle and Lebaron, Philippe
Journal: Applied and environmental microbiology (2002): 3102-7
Authors: Uchihara, T and Nakamura, A and Nagaoka, U and Yamazaki, M and Mori, O
Journal: Histochemistry and cell biology (2000): 447-51
Authors: Webb, D R and Bonfiglioli, R G and Carraro, L and Osler, R and Symons, R H
Journal: Phytopathology (1999): 894-901