iFluor® 610 Styramide
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. iFluor® 610 Styramide is a new unique red fluorescent PSA reagent for multicolor application with our existing PSA and TSA reagents. AAT Bioquest offers the largest collection of TSA regents. We are the exclusive source of the superior PSA reagents for multicolor applications.
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.
Note Prepare the 100X H2O2 solution fresh on the day of use.
1. iFluor™ 610 Styramide stock solution (100X)
Add 100 µL of DMSO into the vial of iFluor™ 610 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.
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™ 610 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
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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
References
View all 12 references: Citation Explorer
Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.
Authors: Jessop, Peter and Gering, Martin
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 193-208
Authors: Jessop, Peter and Gering, Martin
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 193-208
Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation.
Authors: Kuwajima, Masaaki and Ostrovskaya, Olga I and Cao, Guan and Weisberg, Seth A and Harris, Kristen M and Zemelman, Boris V
Journal: PloS one (2020): e0226797
Authors: Kuwajima, Masaaki and Ostrovskaya, Olga I and Cao, Guan and Weisberg, Seth A and Harris, Kristen M and Zemelman, Boris V
Journal: PloS one (2020): e0226797
Intensive Immunofluorescence Staining Methods for Low Expression Protein: Detection of Intestinal Stem Cell Marker LGR5.
Authors: Yamazaki, Masaki and Kato, Atsuhiko and Zaitsu, Yoko and Watanabe, Takeshi and Iimori, Makoto and Funahashi, Shinichi and Kitao, Hiroyuki and Saeki, Hiroshi and Oki, Eiji and Suzuki, Masami
Journal: Acta histochemica et cytochemica (2015): 159-64
Authors: Yamazaki, Masaki and Kato, Atsuhiko and Zaitsu, Yoko and Watanabe, Takeshi and Iimori, Makoto and Funahashi, Shinichi and Kitao, Hiroyuki and Saeki, Hiroshi and Oki, Eiji and Suzuki, Masami
Journal: Acta histochemica et cytochemica (2015): 159-64
Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry.
Authors: Clutter, Matthew R and Heffner, Garrett C and Krutzik, Peter O and Sachen, Kacey L and Nolan, Garry P
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2010): 1020-31
Authors: Clutter, Matthew R and Heffner, Garrett C and Krutzik, Peter O and Sachen, Kacey L and Nolan, Garry P
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2010): 1020-31
Methoxychlor and estradiol induce oxidative stress DNA damage in the mouse ovarian surface epithelium.
Authors: Symonds, Daniel A and Merchenthaler, Istvan and Flaws, Jodi A
Journal: Toxicological sciences : an official journal of the Society of Toxicology (2008): 182-7
Authors: Symonds, Daniel A and Merchenthaler, Istvan and Flaws, Jodi A
Journal: Toxicological sciences : an official journal of the Society of Toxicology (2008): 182-7
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