iFluor 488™ PSA™ Imaging Kit with Goat Anti-Rabbit IgG

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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 Tests 45205 $495


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





Overview

PlatformsFluorescence microscope
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™ 488 PSA kit is a much superior replacement for Alexa Fluor 488 tyramide-based kit or other spectrally similar fluorescent tyramide or TSA kits.




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Protocol


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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

Secondary antibody-HRP

Ex (nm)

Em (nm)

45200

iFluor™ 350 Styramide

100 tests

Goat Anti-Rabbit IgG

345

442

45205

iFluor™ 488 Styramide

100 tests

Goat Anti-Rabbit IgG

491

514

45220

iFluor™ 555 Styramide

100 tests

Goat Anti-Rabbit IgG

552

567

45230

iFluor™ 594 Styramide

100 tests

Goat Anti-Rabbit IgG

592

619

45240

iFluor™ 647 Styramide

100 tests

Goat Anti-Rabbit IgG

649

665

45250

iFluor™ 350 Styramide

100 tests

Goat Anti-Mouse IgG

345

442

45260

iFluor™ 488 Styramide

100 tests

Goat Anti-Mouse IgG

491

514

45270

iFluor™ 555 Styramide

100 tests

Goat Anti-Mouse IgG

552

567

45280

iFluor™ 594 Styramide 100 tests Goat Anti-Mouse IgG 592 619

45290

iFluor™ 647 Styramide 100 tests Goat Anti-Mouse IgG 649 665
Key parameters
Instrument:Fluorescence microscope
Excitation:FITC filter set
Emission:FITC filter set
Recommended plate:Black wall/clear bottom
Instrument specification(s):FITC 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 (Component A) 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. 
  1. Secondary antibody-HRP stock solution (100X):
    Add 100 µL of PBS with 1% BSA into the vial of secondary antibody-HRP (Component D) to prepare the 100X stock solution. Note: This 100x stock solution may be stored at 2-8 oC for up to 3 months. 
  1. H2O2 solution (100X):
    Add 1 mL of 3% hydrogen peroxide (Component E) to 9 mL 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:
    Dilute the 100X secondary antibody-HRP stock solution 1:100 in PBS with 1% BSA. Note: The secondary antibody-HRP provided in this kit is sufficient for 100 tests based on 100 µL HRP working solution per coverslip or per well in a 96-well microplate.
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 oC.

  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.





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Resources
Documents
1. Enzyme Probes & Assay Kits

Certificate of Analysis