logo
AAT Bioquest

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
  1. Fix/permeabilize/block cells or tissue
  2. Add primary antibody in blocking buffer
  3. Add HRP-conjugated secondary antibody
  4. 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™ 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.

Cell fixation and permeabilization
  1. Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
  2. Rinse the cells or tissue with PBS twice.
  3. Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
  4. 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
  1. 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.
  2. Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.
  3. Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.
  4. Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4 °C.
  5. Wash with PBS three times for 5 minutes each.
  6. 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.
  7. Wash with PBS three times for 5 minutes each. 

Styramide labeling
  1. 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.
  2. Rinse with PBS three times. 

Counterstain and fluorescence imaging
  1. 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.
  2. Mount the coverslip using a mounting medium with anti-fading properties.
  3. 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

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 Styramide *Superior Replacement for Alexa Fluor 350 tyramide*3454502000010.9510.830.23
iFluor® 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide and Opal 520*4915167500010.910.210.11
iFluor® 546 Styramide *Superior Replacement for Alexa Fluor 546 tyramide*54155710000010.6710.250.15
iFluor® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570*55757010000010.6410.230.14
iFluor® 568 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*56858710000010.5710.340.15
iFluor® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide*58760320000010.5310.050.04
iFluor® 647 Styramide *Superior Replacement for Alexa Fluor 647 tyramide*65667025000010.2510.030.03
iFluor® 680 Styramide *Superior Replacement for Alexa Fluor 680 tyramide and Opal 690*68470122000010.2310.0970.094
iFluor® 700 Styramide *Superior Replacement for Alexa Fluor 700 tyramide*69071322000010.2310.090.04
iFluor® 750 Styramide *Superior Replacement for Alexa Fluor 750 tyramide*75777927500010.1210.0440.039
iFluor® 790 Styramide *Superior Replacement for Alexa Fluor 790 tyramide*78781225000010.1310.10.09
iFluor® 450 Styramide *Superior Replacement for Opal Polaris 480*4515024000010.8210.450.27
iFluor® 514 Styramide *Superior Replacement for Opal 540*5115277500010.8310.2650.116
iFluor® 532 Styramide5375609000010.6810.260.16
iFluor® 633 Styramide *Superior Replacement for Opal 650*64065425000010.2910.0620.044
iFluor® 440 Styramide4344804000010.6710.3520.229
iFluor® 460 Styramide468493800001~0.810.980.46
iFluor® 660 Styramide66367825000010.2610.070.08
iFluor® 405 Styramide4034273700010.9110.480.77
iFluor® 570 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*55757012000010.581--
iFluor® 670 Styramide *Replacement for Opal 690*67168220000010.5510.030.033
Show More (12)

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

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
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
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
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
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
Page updated on December 1, 2024

Ordering information

Price
Unit size
Catalog Number44904
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Molecular weight

1321.64

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.32

Correction Factor (280 nm)

0.49

Extinction coefficient (cm -1 M -1)

1100001

Excitation (nm)

610

Emission (nm)

628

Quantum yield

0.851

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12171501

Platform

Fluorescence microscope

ExcitationCy3, TRITC filter set
EmissionCy3, TRITC filter set
Recommended plateBlack wall, clear bottom
Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using iFluor® 610 styramide (Cat No. 44904) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using iFluor® 610 styramide (Cat No. 44904) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).
Formalin-fixed, paraffin-embedded (FFPE) human lung tissue was labeled with anti-EpCAM mouse mAb followed by HRP-labeled goat anti-mouse IgG (Cat No. 16728). The fluorescence signal was developed using iFluor® 610 styramide (Cat No. 44904) and detected with a TRITC/Cy3 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).