iFluor™ 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide*

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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.
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Unit Size: Cat No: Price (USD): Qty:
100 Slides 45027 $295


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

Ex/Em (nm)556/569
MW1023.15
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Horseradish Peroxidase (HRP)
Related Vital Stains
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™ 555 Styramide is a superior replacement for Alexa Fluor 555 tyramide or other spectrally similar fluorescent tyramide conjugates or TSA reagents.




Calculators
Common stock solution preparation

Table 1. Volume of needed to reconstitute specific mass of iFluor™ 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Move mouse over grid to display wavelength & intensity values.

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Wavelength (nm)





Protocol


Quick Preview

This protocol only provides a guideline, and should be modified according to your specific needs.
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

Cat. #

Product Name

Unit

Ex (nm)

Em (nm)

45000

iFluor™ 350 Styramide

100 slides

345

442

45020

iFluor™ 488 Styramide

100 slides

491

514

45025

iFluor™ 546 Styramide

100 slides

541

557

45027

iFluor™ 555 Styramide

100 slides

552

567

45030

iFluor™ 568 Styramide

100 slides

568

587

45035

iFluor™ 594 Styramide

100 slides

592

619

45045

iFluor™ 647 Styramide

100 slides

649

665

45050

iFluor™ 680 Styramide

100 slides

676

695

45055

iFluor™ 700 Styramide

100 slides

685

710

45065

iFluor™ 750 Styramide

100 slides

749

775

45070

iFluor™ 790 Styramide 100 slides 782 811

45300

Biotin Styramide 100 slides    
45305 DIG Styramide 100 slides    
45310 DNP Styramide 100 slides    
Key parameters
Instrument:Fluorescence microscope
Excitation:Cy3/TRITC filter set
Emission:Cy3/TRITC filter set
Recommended plate:Black wall/clear bottom
Instrument specification(s):Cy3/TRITC filter set
Preparation of stock solution
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. Styramide™ stock solution (100X):
    Add 100 µL of DMSO into the vial of iFluor™ dye-labeled 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 and avoid repeat freeze-thaw cycles.

  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. 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

Example data analysis and figures

Figure 1. 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.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.









Resources
 
Safety Data Sheet (SDS)


Documents
1. Enzyme Probes & Assay Kits

Certificate of Analysis