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

Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma using two imaging signal amplification kits. Tissue sections were stained with rabbit anti-EpCam antibody and then followed PSA™ method using iFluor® 488 PSA™ Imaging Kit with goat anti-rabbit IgG (Cat#45205) or Thermo Fisher Alexa Fluor® 488 Superboost Kit  respectively. Cell nucleus were stained with Nuclear Blue™ DCS1 (Cat#17548).

Sequential immunostaining of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma using 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.

Sensitivity of Styramide™ Super Signal Amplification Kits. (A) HeLa cells were fixed, permeabilized and labeled with various concentrations of rabbit anti-Tubulin primary antibody. The manufacturer recommendation was 1:500 dilution. Cells were then stained with Goat anti-Rabbit IgG secondary antibody directly conjugated with Alexa Fluor® 488, or by amplified methods using a HRP-labeled Goat anti-Rabbit IgG secondary antibody followed by Alexa Fluor® 488 tyramide or iFluor® 488 Styramide™ (Cat#45205), respectively. Fluorescence images were taken using the FITC filter set and analyzed with the same exposure time. (B) Relative fluorescence signal intensity was measured and compared between different detection methods.

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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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Spectral properties

Correction Factor (260 nm)	0.21
Correction Factor (280 nm)	0.11
Extinction coefficient (cm ^-1 M ^-1)	75000¹
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.9¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.11

Extinction coefficient (cm ^-1 M ^-1)

75000¹

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.9¹

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.

Platform

Fluorescence microscope

Excitation	FITC filter set
Emission	FITC filter set
Recommended plate	Black wall/clear bottom
Instrument specification(s)	FITC filter set

Components

Example protocol

AT A GLANCE

Protocol Summary

Fix/permeabilize/block cells or tissue
Add primary antibody in blocking buffer
Add HRP-conjugated secondary antibody
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™ 488-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.

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

Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
Rinse the cells or tissue with PBS twice.
Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
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

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.
Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.
Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.
Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at 4 °C.
Wash with PBS three times for 5 minutes each.
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.
Wash with PBS three times for 5 minutes each.

Styramide labeling

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.
Rinse with PBS three times.

Counterstain and fluorescence imaging

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.
Mount the coverslip using a mounting medium with anti-fading properties.
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

Spectral properties

Correction Factor (260 nm)	0.21
Correction Factor (280 nm)	0.11
Extinction coefficient (cm ^-1 M ^-1)	75000¹
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.9¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
iFluor® 350 PSA™ Imaging Kit with Goat Anti-Rabbit IgG	345	450	20000¹	0.95¹	0.83	0.23
iFluor® 555 PSA™ Imaging Kit with Goat Anti-Rabbit IgG	557	570	100000¹	0.64¹	0.23	0.14
iFluor® 594 PSA™ Imaging Kit with Goat Anti-Rabbit IgG	588	604	180000¹	0.53¹	0.05	0.04
iFluor® 647 PSA™ Imaging Kit with Goat Anti-Rabbit IgG	656	670	250000¹	0.25¹	0.03	0.03
iFluor 488™ PSA™ Imaging Kit with Goat Anti-Mouse IgG	491	516	75000¹	0.9¹	0.21	0.11

Images

Figure 1. Fluorescence IHC of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma using two imaging signal amplification kits. Tissue sections were stained with rabbit anti-EpCam antibody and then followed PSA™ method using iFluor® 488 PSA™ Imaging Kit with goat anti-rabbit IgG (Cat#45205) or Thermo Fisher Alexa Fluor® 488 Superboost Kit respectively. Cell nucleus were stained with Nuclear Blue™ DCS1 (Cat#17548).

Figure 2. Sequential immunostaining of formaldehyde-fixed, paraffin-embedded human lung adenocarcinoma using 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.

Figure 3. Sensitivity of Styramide™ Super Signal Amplification Kits. (A) HeLa cells were fixed, permeabilized and labeled with various concentrations of rabbit anti-Tubulin primary antibody. The manufacturer recommendation was 1:500 dilution. Cells were then stained with Goat anti-Rabbit IgG secondary antibody directly conjugated with Alexa Fluor® 488, or by amplified methods using a HRP-labeled Goat anti-Rabbit IgG secondary antibody followed by Alexa Fluor® 488 tyramide or iFluor® 488 Styramide™ (Cat#45205), respectively. Fluorescence images were taken using the FITC filter set and analyzed with the same exposure time. (B) Relative fluorescence signal intensity was measured and compared between different detection methods.

Figure 4. 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.