logo
AAT Bioquest

iFluor® 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide and Opal 520*

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® 488 Styramide is a superior replacement for Alexa Fluor 488 tyramide, Opal 520 or other spectrally similar fluorescent tyramide conjugates or TSA reagents.

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® 488 Styramide conjugate to make 100X Styramide stock solution.

Note: Make single-use aliquots and store unused 100X stock solution at 2-8 °C, protected from light. 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

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide and Opal 520* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM96.34 µL481.7 µL963.4 µL4.817 mL9.634 mL
5 mM19.268 µL96.34 µL192.68 µL963.4 µL1.927 mL
10 mM9.634 µL48.17 µL96.34 µL481.7 µL963.4 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 488 maleimide4915167500010.910.210.11
iFluor® 488 amine4915167500010.910.210.11
iFluor® 488 hydrazide4915167500010.910.210.11
iFluor® 488 tyramide4915167500010.910.210.11
iFluor® 488 azide4915167500010.910.210.11
iFluor® 488 alkyne4915167500010.910.210.11
iFluor® 350 Styramide *Superior Replacement for Alexa Fluor 350 tyramide*3454502000010.9510.830.23
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® 488 TCO4915167500010.910.210.11
iFluor® 488 Tetrazine4915167500010.910.210.11
iFluor®488-dUTP *1 mM in TE Buffer (pH 7.5)*4915167500010.910.210.11
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® 570 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*55757012000010.581--
iFluor® 670 Styramide *Replacement for Opal 690*67168220000010.5510.030.033
Show More (21)

Citations

View all 3 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
Inhibition of platelet-derived growth factor pathway suppresses tubulointerstitial injury in renal congestion
Authors: Matsuki, Takuma and Hirose, Takuo and Ohsaki, Yusuke and Shimada, Satoshi and Endo, Akari and Ito, Hiroki and Takahashi, Chika and Yamakoshi, Seiko and Oba-Yabana, Ikuko and Anan, Go and others,
Journal: Journal of hypertension (2022): 1935--1949
CD95/Fas protects triple negative breast cancer from anti-tumor activity of NK cells
Authors: Qadir, Abdul S and Gu{\'e}gan, Jean Philippe and Ginestier, Christophe and Chaibi, Assia and Bessede, Alban and Charafe-Jauffret, Emmanuelle and Macario, Manon and Lavou{\'e}, Vincent and de la Motte Rouge, Thibault and Law, Calvin and others,
Journal: Iscience (2021): 103348

References

View all 50 references: Citation Explorer
Immunofluorescent Staining of Adult Murine Paraffin-Embedded Skeletal Tissue.
Authors: Felsenthal, Neta and Zelzer, Elazar
Journal: Methods in molecular biology (Clifton, N.J.) (2021): 337-344
Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide.
Authors: Xiao, Lu and Labaer, Joshua and Guo, Jia
Journal: Cells (2021)
Single-cell RNA sequencing of human liver reveals hepatic stellate cell heterogeneity.
Authors: Payen, Valéry L and Lavergne, Arnaud and Alevra Sarika, Niki and Colonval, Megan and Karim, Latifa and Deckers, Manon and Najimi, Mustapha and Coppieters, Wouter and Charloteaux, Benoît and Sokal, Etienne M and El Taghdouini, Adil
Journal: JHEP reports : innovation in hepatology (2021): 100278
Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide.
Authors: Pham, Thai and Liao, Renjie and Labaer, Joshua and Guo, Jia
Journal: Molecules (Basel, Switzerland) (2021)
Accessibility-dependent topology studies of membrane proteins using a SpyTag/SpyCatcher protein-ligation system.
Authors: Bae, Yoonji and Lee, Sang Kwon and Chae, Young Chan and Park, Chan Young and Kang, Sebyung
Journal: International journal of biological macromolecules (2021): 171-178
Page updated on December 11, 2024

Ordering information

Price
Unit size
Catalog Number45020
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

1037.99

Solvent

DMSO

Spectral properties

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.11

Extinction coefficient (cm -1 M -1)

750001

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.91

Storage, safety and handling

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

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall, clear bottom
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA<strong>&nbsp;&trade;&nbsp;</strong>&nbsp;and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with&nbsp;rabbit anti-EpCam antibody and then incubated with polyHRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 488 Styramide&trade; (Cat#45020) or Alexa Fluor&reg; 488 tyramide stain respectively. &nbsp;Images showed that iFluor 488&trade; PSA&trade; super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor&reg; 488 TSA method.
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA<strong>&nbsp;&trade;&nbsp;</strong>&nbsp;and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with&nbsp;rabbit anti-EpCam antibody and then incubated with polyHRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 488 Styramide&trade; (Cat#45020) or Alexa Fluor&reg; 488 tyramide stain respectively. &nbsp;Images showed that iFluor 488&trade; PSA&trade; super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor&reg; 488 TSA method.
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma positive tissue using PSA<strong>&nbsp;&trade;&nbsp;</strong>&nbsp;and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with&nbsp;rabbit anti-EpCam antibody and then incubated with polyHRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 488 Styramide&trade; (Cat#45020) or Alexa Fluor&reg; 488 tyramide stain respectively. &nbsp;Images showed that iFluor 488&trade; PSA&trade; super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor&reg; 488 TSA method.
Sensitivity of iFluor® 488 Styramide (A) HeLa cells were fixed, permeabilized and labeled with various concentrations of rabbit anti-Tubulin primary antibody. The manufacturer recommendation was 1:500 dilution. Cells were then stained with Goat anti-Rabbit IgG secondary antibody directly conjugated with Alexa Fluor&reg; 488, or by amplified methods using a HRP-labeled Goat anti-Rabbit IgG secondary antibody followed by Alexa Fluor&reg; 488 tyramide or iFluor® 488 Styramide&trade; (Cat#45020), respectively. Fluorescence images were taken using the FITC filter set and analyzed with the same exposure time. (B) Relative fluorescence signal intensity was measured and compared between different detection methods.
Power Styramide&trade; Signal Amplification (PSA&trade;) 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&trade; 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.&nbsp; PSA radicals have much higher reactivity than tyramide radicals, making the PSA system much faster, more robust and sensitive than the traditional TSA reagents.