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iFluor® 670 Styramide *Replacement for Opal 690*

The Power Styramide™ Signal Amplification (PSA™) system is a highly sensitive method for detecting low-abundant targets in fluorescent immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (ISH). When combined with bright and photostable iFluor® dyes, Styramide™ conjugates offer sharper and more precise results, exceeding the sensitivity of standard ICC/IHC/ISH methods by over 100 times, all while reducing the consumption of primary antibodies. Like tyramide signal amplification (TSA), PSA™ also uses the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ. However, Styramide™ radicals are more reactive than tyramide radicals, making the labeling of the target protein or nucleic acid sequence faster and more robust. This provides fluorescence signals that are 10-50 times greater than those generated by tyramide (TSA) reagents. iFluor® 670 Styramide™ uses the bright red-fluorescent dye iFluor® 670 (Ex/Em = 671/682 nm) to label targets in situ. The high fluorescence quantum yield and photostability of iFluor® 670 styramide outperforms Opal 690 tyramide and other spectrally similar reagents, making it a superior alternative for imaging 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

Styramide™ stock solution (100X)

Add 100 µL of DMSO into the vial of iFluor® dye-labeled Styramide™ conjugate to make a 100X Styramide™ stock solution.

Note: Make single-use aliquots, and store unused 100X stock solution at 2-8 °C in a dark place and avoid repeat freeze-thaw cycles.

Hydrogen peroxide stock solution (100X)

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

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.

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 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

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 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.

  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.

    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.

  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® 610 Styramide61062811000010.8510.320.49
iFluor® 660 Styramide66367825000010.2610.070.08
iFluor® 405 Styramide4034273700010.9110.480.77
iFluor® 670 maleimide67168220000010.5510.030.033
iFluor® 570 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*55757012000010.581--
iFluor® 670 acid67168220000010.5510.030.033
Show More (14)

Citations

View all 2 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
Protocol for detection of glial complement expression in relation to amyloid plaques in mouse brain with combined FISH and IHC
Authors: Rao, Sadhna and Yung, Joanna and Edick, Melissa Gonzalez and Hanson, Jesse E
Journal: STAR protocols (2024): 103388

References

View all 6 references: Citation Explorer
RNA methylation-driven assembly of fluorescence-encoded nanostructures for sensitive detection of m6A modification writer METTL3/14 complex in human breast tissues.
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
Quantum dot-based FRET for sensitive determination of hydrogen peroxide and glucose using tyramide reaction.
Authors: Huang, Xiangyi and Wang, Jinjie and Liu, Heng and Lan, Tao and Ren, Jicun
Journal: Talanta (2013): 79-84
Dual enhancement of triple immunofluorescence using two antibodies from the same species.
Authors: Nakamura, Ayako and Uchihara, Toshiki
Journal: Journal of neuroscience methods (2004): 67-70
Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry.
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
Dual enhancement of double immunofluorescent signals by CARD: participation of ubiquitin during formation of neurofibrillary tangles.
Authors: Uchihara, T and Nakamura, A and Nagaoka, U and Yamazaki, M and Mori, O
Journal: Histochemistry and cell biology (2000): 447-51
Page updated on December 17, 2024

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Catalog Number45047
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Physical properties

Molecular weight

1114.43

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.03

Correction Factor (280 nm)

0.033

Extinction coefficient (cm -1 M -1)

2000001

Excitation (nm)

671

Emission (nm)

682

Quantum yield

0.551

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

ExcitationCy5 filter set
EmissionCy5 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® 670 styramide (Cat No. 45047) and detected with a Cy5 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® 670 styramide (Cat No. 45047) and detected with a Cy5 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® 670 styramide (Cat No. 45047) and detected with a Cy5 filter set. Nuclei (blue) were counterstained with DAPI (Cat No. 17507).