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iFluor 555™ PSA™ Imaging Kit with Goat Anti-Mouse IgG

Fluorescence IHC for HER2/ErbB2 detection in formaldehyde-fixed paraffin-embedded breast cancer tissue. Human Her2/Neu (c-erbB-2) positive tissue sections were incubated with Rabbit mAb HER2/ErbB2 diluted 1:5000 (Top) or 1:250 (Bottom), respectively. The tissue was then incubated with HRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor™ 555 Styramide™ (Top) or Alexa Fluor® 555 tyramide (Bottom), respectively. Compared to tyramide amplification, the increase in sensitivity of Styramide™ super signal amplification allows the use of fewer primary antibodies. The same level of sensitivity can be achieved with a significant reduction in the amount of target antibody used (10-50 fold). The cell nucleus was costained with DAPI (Cat No. 17507).
Fluorescence IHC for HER2/ErbB2 detection in formaldehyde-fixed paraffin-embedded breast cancer tissue. Human Her2/Neu (c-erbB-2) positive tissue sections were incubated with Rabbit mAb HER2/ErbB2 diluted 1:5000 (Top) or 1:250 (Bottom), respectively. The tissue was then incubated with HRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor™ 555 Styramide™ (Top) or Alexa Fluor® 555 tyramide (Bottom), respectively. Compared to tyramide amplification, the increase in sensitivity of Styramide™ super signal amplification allows the use of fewer primary antibodies. The same level of sensitivity can be achieved with a significant reduction in the amount of target antibody used (10-50 fold). The cell nucleus was costained with DAPI (Cat No. 17507).
Sequential immunostaining of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma using two iFluor™ PSA™ Imaging kits. EpCam were labeled with rabbit anti-EpCam antibodies and iFluor™ 488 PSA™ Imaging Kit with goat anti-rabbit IgG, followed by washing. Pan-Keratin were labeled with mouse anti-pan Keratin antibodies and iFluor™ 555 PSA™ Imaging Kit with goat anti-mouse IgG. Nuclei were labeled with DAPI. Images were acquired on a confocal microscope.
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded using PSA<strong> ™ </strong> and TSA amplified methods. Human lung adenocarcinoma positive tissue sections were stained with mouse anti-EpCam antibody and then followed by PSA™ method using iFluor 555™ PSA™ Imaging Kit with Goat Anti-Mouse IgG (Cat#45270) or TSA method using  Alexa Fluor® 555 tyramide  respectively.  Images showed that PSA™ super signal amplification can increase the sensitivity of fluorescence IHC over Alexa Fluor® 555 TSA method. Cell nucleus were stained with Nuclear Blue™ DCS1 (Cat#17548).
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.
Ordering information
Price ()
Catalog Number45270
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
Spectral properties
Correction Factor (260 nm)0.23
Correction Factor (280 nm)0.14
Extinction coefficient (cm -1 M -1)1000001
Excitation (nm)557
Emission (nm)570
Quantum yield0.641
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
0.23
Correction Factor (280 nm)
0.14
Extinction coefficient (cm -1 M -1)
1000001
Excitation (nm)
557
Emission (nm)
570
Quantum yield
0.641
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 PSA kit is a much superior replacement for Alexa Fluor 555 tyramide-based kit or other spectrally similar fluorescent tyramide or TSA kits.

Platform


Fluorescence microscope

ExcitationCy3/TRITC filter set
EmissionCy3/TRITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)Cy3/TRITC filter set

Components


Component A: iFluor™ 555 Styramide™ conjugate1 vial (lyophilized powder)
Component B: Styramide™ Reaction Buffer 1 bottle (10 mL)
Component C: DMSO1 vial (100 µL)
Component D: Secondary Antibody-HRP (Goat Anti-Mouse IgG-HRP)1 vial (100 µL) (100X)
Component E: Stabilized 3% Hydrogen Peroxide (H2O2)1 bottle (11 mL)

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. Styramide™ stock solution (100X)
Add 100 µL of DMSO into the vial of iFluor™ 555-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 dark place and avoid repeat freeze-thaw cycles.


2. 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-tissueimmunohistochemistry-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)0.23
Correction Factor (280 nm)0.14
Extinction coefficient (cm -1 M -1)1000001
Excitation (nm)557
Emission (nm)570
Quantum yield0.641

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor 555™ PSA™ Imaging Kit with Goat Anti-Rabbit IgG55757010000010.6410.230.14
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 594™ PSA™ Imaging Kit with Goat Anti-Mouse IgG58860418000010.5310.050.04
iFluor 647™ PSA™ Imaging Kit with Goat Anti-Mouse IgG65667025000010.2510.030.03