DIG Styramide *Superior Replacement for DIG tyramide*
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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 | 840.07 |
Solvent | DMSO |
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 | 12352200 |
Overview | SDSProtocol |
Molecular weight 840.07 |
Example protocol
AT A GLANCE
- 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
Add 100 µL of DMSO into the vial of DIG-labeled Styramide™ conjugate to make 100X Styramide™ stock solution.
Note: Make single-use aliquots and store unused 100X stock solution at -20 °C in a dark place. Avoid repeat 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
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.
Make appropriate concentration of secondary antibody-HRP working solution as per the manufacturer's recommendations.
Make appropriate concentration of Anti-DIG antibody conjugate working solution as per the manufacturer's recommendations.
SAMPLE EXPERIMENTAL PROTOCOL
This protocol is applicable for both cells and tissues staining.
- 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.
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
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 with a 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.
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 a 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 a lower concentration of Styramide™ in the working solution.- Rinse with PBS three times.
- Apply 100 µL of secondary Anti-DIG antibody conjugate working solution to each sample and incubate for 60 minutes at room temperature.
- Wash with PBS three times for 5 minutes each.
- 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 Styramide 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
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 119.038 µL | 595.188 µL | 1.19 mL | 5.952 mL | 11.904 mL |
5 mM | 23.808 µL | 119.038 µL | 238.075 µL | 1.19 mL | 2.381 mL |
10 mM | 11.904 µL | 59.519 µL | 119.038 µL | 595.188 µL | 1.19 mL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Images
Citations
Authors: Zhang, Jing and Liu, Hong-li and Liu, Jing-bo and Zhang, Yuan and Liu, Jing and Li, Yan-hua
Journal: Biochemical and Biophysical Research Communications (2021): 95--102
Application notes
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