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iFluor® 594 PSA™ Imaging Kit with Goat Anti-Mouse IgG

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® 594 PSA kit is a much superior replacement for Alexa Fluor 594 tyramide-based kit or other spectrally similar fluorescent tyramide or TSA kits.

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)
  1. Add 100 µL of DMSO into the vial of iFluor® 594-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 °C in a dark place. Avoid repeat freeze-thaw cycles.

Hydrogen peroxide solution (100X)
  1. 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

Styramide working solution (1X)
  1. 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
  1. 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 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.
Multiplex with primary antibodies from different species

Important: When performing multiplexing experiments with PSA™ Imaging Kits, it is essential to maintain a specific imaging sequence. To ensure clear and accurate results, it is recommended to image the targets from least to most abundant, with the nucleus counterstained last. Following this optimized imaging sequence will enhance the precision and quality of your experiment.

  1. Following Step 2 in the Styramide labeling section, you can multiplex cells or tissue samples either by employing another iFluor® PSA™ Imaging Kit or by utilizing conventional IHC/ICC protocols.

  2. For multiplexing, employ a primary antibody from a host different from the one utilized in Step 4 of the Peroxidase labeling section, along with a fluorescent label that is spectrally compatible with the initial fluorescent label.

Multiplex with primary antibodies from the same species in IHC
  1. Dilute 10X Citrate Buffer (pH 6.0) (Cat. No. 10000) 1:10 in distilled water.

  2. Following Step 2 of the Styramide labeling process, immerse the tissue in a diluted citrate buffer (pH 6.0) and heat in a microwave oven at 100% power until boiling (1–2.5 minutes).

  3. Reduce the power to 20% and microwave for an additional 15 minutes.

  4. Allow the tissue sample to cool to room temperature while it remains in the citrate buffer.

  5. Wash the sample twice with 1X PBS. Then, repeat the Peroxidase labeling and Styramide labeling steps using a primary antibody of the same species, if desired.

  6. Use an iFluor® PSA™ Imaging Kit that is spectrally compatible with the iFluor® 350 PSA™ Imaging Kit used in the first round.

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 instructions 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. Recommended nucleus counterstains.

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® 594 PSA™ Imaging Kit with Goat Anti-Rabbit IgG58760320000010.5310.050.04
iFluor® 350 PSA™ Imaging Kit with Goat Anti-Mouse IgG3454502000010.9510.830.23
iFluor® 488 PSA™ Imaging Kit with Goat Anti-Mouse IgG4915167500010.910.210.11
iFluor® 555 PSA™ Imaging Kit with Goat Anti-Mouse IgG55757010000010.6410.230.14
iFluor® 647 PSA™ Imaging Kit with Goat Anti-Mouse IgG65667025000010.2510.030.03

Citations

View all 1 citations: Citation Explorer
OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules
Authors: Nakanishi, Shinobu and Hasegawa, Tatsuya and Maeno, Katsuyuki and Motoyama, Akira and Denda, Mitsuhiro
Journal: iScience (2024)
Page updated on November 7, 2024

Ordering information

Price
Unit size
Catalog Number45280
Quantity
Add to cart

Additional ordering information

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Spectral properties

Absorbance (nm)

587

Correction Factor (260 nm)

0.05

Correction Factor (280 nm)

0.04

Extinction coefficient (cm -1 M -1)

2000001

Excitation (nm)

587

Emission (nm)

603

Quantum yield

0.531

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationCy3, TRITC filter set
EmissionCy3, TRITC filter set
Recommended plateBlack wall, clear bottom

Components

Fluorescence IHC of formaldehyde-fixed, paraffin-embedded using PSA<strong> &trade; </strong>&nbsp;and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with mouse anti-EpCam antibody and then followed by PSA&trade;&nbsp;method using iFluor 594&trade; PSA&trade; Imaging Kit with Goat Anti-Mouse IgG (Cat#45280) or TSA method using&nbsp; Alexa Fluor&reg; 594 tyramide&nbsp; respectively.&nbsp; Images showed that PSA&trade; super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor&reg; 594 TSA method. Cell nucleus were stained with Nuclear Blue&trade; DCS1 (Cat#17548).
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded using PSA<strong> &trade; </strong>&nbsp;and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with mouse anti-EpCam antibody and then followed by PSA&trade;&nbsp;method using iFluor 594&trade; PSA&trade; Imaging Kit with Goat Anti-Mouse IgG (Cat#45280) or TSA method using&nbsp; Alexa Fluor&reg; 594 tyramide&nbsp; respectively.&nbsp; Images showed that PSA&trade; super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor&reg; 594 TSA method. Cell nucleus were stained with Nuclear Blue&trade; DCS1 (Cat#17548).
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded using PSA<strong> &trade; </strong>&nbsp;and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with mouse anti-EpCam antibody and then followed by PSA&trade;&nbsp;method using iFluor 594&trade; PSA&trade; Imaging Kit with Goat Anti-Mouse IgG (Cat#45280) or TSA method using&nbsp; Alexa Fluor&reg; 594 tyramide&nbsp; respectively.&nbsp; Images showed that PSA&trade; super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor&reg; 594 TSA method. Cell nucleus were stained with Nuclear Blue&trade; DCS1 (Cat#17548).
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.