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iFluor® 633 Styramide *Superior Replacement for Opal 650*
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 florescence 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 covalent deposition of fluorophores in situ. PSA radicals have much higher reactivity than tyramide radicals, making the PSA system much faster, more robust and sensitive than the traditional TSA reagents. Compared to tyramide reagents, the Styramide™ conjugates have ability to label the target at higher efficiency and thus generate significantly higher fluorescence signal. Styramide™ conjugates also allow significantly less consumption of primary antibody compared to standard directly conjugate method or tyramide amplification with the same level of sensitivity. iFluor® 633 Styramide is optimized to a superior replacement for Opal Polaris 650 or other spectrally similar fluorescent tyramide conjugates or TSA reagents.
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.
Note Make single use aliquots, and store unused 100X stock solution at 2-8 oC in dark place and avoid repeat freeze-thaw cycles.
Note Prepare the 100X H2O2 solution fresh on the day of use.
1. iFluor™ 633 Styramide stock solution (100X)
Add 100 µL of DMSO into the vial of iFluor™ 633 Styramide conjugate to make 100X Styramide stock solution.Note Make single use aliquots, and store unused 100X stock solution at 2-8 oC in dark place and avoid repeat freeze-thaw cycles.
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™ 633 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 |
Spectrum
Product family
Citations
View all 1 citations: Citation Explorer
Membrane progesterone receptor $\gamma$ (paqr5b) is essential for the formation of neurons in the zebrafish olfactory rosette
Authors: Mustary, Umme Habiba and Maeno, Akiteru and Rahaman, Md Mostafizur and Ali, Md Hasan and Tokumoto, Toshinobu
Journal: Scientific Reports (2024): 24354
Authors: Mustary, Umme Habiba and Maeno, Akiteru and Rahaman, Md Mostafizur and Ali, Md Hasan and Tokumoto, Toshinobu
Journal: Scientific Reports (2024): 24354
Page updated on December 17, 2024
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