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iFluor® 405 Styramide

Fixed and permeabilized HeLa cells were incubated with rabbit anti-tubulin antibody, then labeled with HRP-labeled Goat anti-Rabbit IgG (Cat No. 16793), and detected using iFluor® 405 styramide (Cat No. 44908). Images were captured on a fluorescence microscope equipped with a DAPI filter set.
Fixed and permeabilized HeLa cells were incubated with rabbit anti-tubulin antibody, then labeled with HRP-labeled Goat anti-Rabbit IgG (Cat No. 16793), and detected using iFluor® 405 styramide (Cat No. 44908). Images were captured on a fluorescence microscope equipped with a DAPI filter set.
Fluorescence images of HeLa cells labeled with rabbit anti-Tubulin primary antibody. Cells were then stained with a HRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 405 Styramide. Fluorescence images were taken using the DAPI filter set.
Ordering information
Price ()
Catalog Number44908
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.48
Correction Factor (280 nm)0.77
Extinction coefficient (cm -1 M -1)370001
Excitation (nm)403
Emission (nm)427
Quantum yield0.911
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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iFluor® 700 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 750 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 790 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 350 goat anti-rabbit IgG (H+L)
iFluor® 405 goat anti-rabbit IgG (H+L)
iFluor® 488 goat anti-rabbit IgG (H+L)
iFluor® 514 goat anti-rabbit IgG (H+L)
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iFluor® 594 goat anti-rabbit IgG (H+L)
iFluor® 633 goat anti-rabbit IgG (H+L)
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iFluor® 680 goat anti-rabbit IgG (H+L)
iFluor® 700 goat anti-rabbit IgG (H+L)
iFluor® 750 goat anti-rabbit IgG (H+L)
iFluor® 790 goat anti-rabbit IgG (H+L)
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iFluor® 488 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 514 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 532 succinimidyl ester
iFluor® 555 succinimidyl ester
iFluor® 594 succinimidyl ester
iFluor® 633 succinimidyl ester
iFluor® 647 succinimidyl ester
iFluor® 660 succinimidyl ester
iFluor® 680 succinimidyl ester
iFluor® 700 succinimidyl ester
iFluor® 750 succinimidyl ester
iFluor® 610 succinimidyl ester
iFluor® 710 succinimidyl ester
iFluor® 790 succinimidyl ester
iFluor® 800 succinimidyl ester
iFluor® 810 succinimidyl ester
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iFluor® 430 succinimidyl ester
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iFluor® 840 succinimidyl ester
iFluor® 560 succinimidyl ester
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iFluor® 460 succinimidyl ester
iFluor® 440 succinimidyl ester
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iFluor® 690 succinimidyl ester
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iFluor® 740 succinimidyl ester
iFluor® 597 succinimidyl ester
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Show More (242)

OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
0.48
Correction Factor (280 nm)
0.77
Extinction coefficient (cm -1 M -1)
370001
Excitation (nm)
403
Emission (nm)
427
Quantum yield
0.911
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. 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® 405 Styramide is a new unique blue fluorescent PSA reagent for multicolor application with our existing PSA and TSA reagents. It has the maximum fluorescence ~421 nm. iFluor® 405 Styramide is a superior replacement for Alexa Fluor 405 tyramide or other spectrally similar fluorescent tyramide conjugates or TSA reagents. AAT Bioquest offers the largest collection of TSA regents. We are the exclusive source of the superior PSA reagents for multicolor applications.

Platform


Fluorescence microscope

ExcitationDAPI/Violet filter set
EmissionDAPI/Violet filter set
Recommended plateBlack wall/clear bottom

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™ 405 Styramide stock solution (100X)
Add 100 µL of DMSO into the vial of iFluor™ 405 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™ 405 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.48
Correction Factor (280 nm)0.77
Extinction coefficient (cm -1 M -1)370001
Excitation (nm)403
Emission (nm)427
Quantum yield0.911

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 405 amine4034273700010.9110.480.77
iFluor® 405 hydrazide4034273700010.9110.480.77
iFluor® 405 maleimide4034273700010.9110.480.77
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*58860418000010.5310.050.04
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 azide4034273700010.9110.480.77
Show More (24)

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
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
Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.
Authors: Wilbanks, Brandon and Garcia, Brian and Byrne, Shane and Dedon, Peter and Maher, L James
Journal: Chembiochem : a European journal of chemical biology (2021): 1400-1404
Adoptive cell therapy of triple negative breast cancer with redirected cytokine-induced killer cells.
Authors: Sommaggio, Roberta and Cappuzzello, Elisa and Dalla Pietà, Anna and Tosi, Anna and Palmerini, Pierangela and Carpanese, Debora and Nicolè, Lorenzo and Rosato, Antonio
Journal: Oncoimmunology (2020): 1777046
A simple, real-time assay of horseradish peroxidase using biolayer interferometry.
Authors: Kojima, Takaaki and Nakane, Ayako and Zhu, Bo and Alfi, Almasul and Nakano, Hideo
Journal: Bioscience, biotechnology, and biochemistry (2019): 1822-1828
The EMARS Reaction for Proximity Labeling.
Authors: Honke, Koichi and Miyagawa-Yamaguchi, Arisa and Kotani, Norihiro
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 1-12