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Power Styramide™ Signal Amplification (PSA™)

Power Styramide™ Signal Amplification (PSA™) is a novel enzymatic amplification method used to detect low-abundance targets in cells and tissues. By combining the superior brightness and photostability of iFluor™ dyes with poly-HRP mediated styramide amplification, PSA™ imaging generates bright fluorescence signals with significantly higher precision and sensitivity (more than 100-fold greater) than conventional immunohistochemistry, immunocytochemistry, and in situ hybridization techniques.

 



Power Styramide™ Signal Amplification



Schematic representation of PSA™ detection method applied to immunolabeling of a target antigen. Using a conventional detection method, cells or tissue samples are probed with an unlabeled primary antibody and an HRP-secondary conjugate. HRP catalyzes the conversion of labeled Styramide™ into highly-reactive Styramide™ radicals that covalently bind to tyrosine residues on and proximal to the enzyme site.
Similar to tyramide signal amplification (TSA), PSA™ imaging uses the analyte-dependent reporter enzyme, horseradish peroxidase (HRP), to catalyze the covalent deposition and binding of labeled-Styramide™ substrates onto a target protein or nucleic acid sequence in situ. In the presence of hydrogen peroxide (H2O2), HRP converts labeled Styramide™ substrates into highly-reactive, short-lived Styramide™ radicals that rapidly bind to tyrosine residues on and proximal to the enzyme site. Styramide™ radicals have much higher reactivity than tyramide radicals, making imaging with PSA™ significantly faster, more robust, and sensitive than conventional TSA labeling. Since the added labeled-Styramide™ are deposited close to the HRP-target site, there is a minimal diffusion-related loss of resolution. PSA™ imaging technology can be readily added to any application that allows for integrating HRP into its protocol. Such applications include IHC, ICC, IF, in situ hybridization, and ELISA.

Advantages of PSA™ Imaging System


Power Styramide™ signal amplification resulting from the rapid catalyzation and covalent deposition of multiple Styramide™ substrates per HRP label translates to many practical benefits, including simplicity, enhanced sensitivity and specificity, and compatibility with other techniques. The higher reactivity of Styramide™ radicals permits more robust and expeditious labeling of Styramide™ at the HRP-target interaction site resulting in stronger signal intensity and better spatial resolution than TSA.

Key Features of PSA™:

  • Ultra-sensitive detection of low-abundance targets, 100-fold greater than IHC, ICC, and IF methods
  • High fluorescence intensity, 10 to 50-fold greater than tyramide
  • Compatible with other fluorescent markers, staining techniques, and PSA™ imaging kits for multiplex analysis
  • Higher reactivity of PSA™ radicals for faster results and equivalent sensitivity and resolution versus radiometric detection
  • Conserve precious antibodies, PSA™ labeling achieves equivalent levels of sensitivity with a significant reduction in primary antibody
  • PSA™ imaging kits are easy-to-use and provide sufficient reagents for 100 tests

iFluor™ 594 Styramide™
Alexa Fluor® 594 tyramide

Fluorescence IHC of formaldehyde-fixed, paraffin-embedded using PSA™ 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 594™ PSA™ Imaging Kit with Goat Anti-Mouse IgG (Cat No. 45290) or TSA method using Alexa Fluor® 594 tyramide, respectively. Images were taken using the TRITC filter set and under the same exposure time. Nuclei were counterstained with Nuclear Blue™ DCS1 (Cat No. 17548).

 

Superior Detection Sensitivity


In immunological staining applications, sensitivity enhancements derived from PSA™ imaging allows for increases in primary antibody dilutions with no sacrifice in assay sensitivity. Furthermore, increasing the primary antibody dilutions reduces nonspecific background signals and overcomes insufficient immunolabeling caused by poor fixation procedures or low-levels of target expression.



Sensitivity of Power Styramide™ Signal Amplification (PSA™) Kits. HeLa cells were fixed, permeabilized, and labeled with various concentrations of rabbit anti-tubulin primary antibody. The manufacturer's recommendation was 1:500 dilution or 2 µg/ml. Cells were then stained with reagents in our iFluor™ 488 PSA™ Imaging Kit with Goat Anti-Rabbit IgG, an Alexa Fluor® 488-labeled tyramide, or an Alexa Fluor® 488-labeled goat anti-rabbit IgG. Cell images were captured from each treatment under the same conditions (using a FITC filter set and analyzed with the same exposure time). Relative fluorescence signal intensity was measured and compared between different detection methods.

 

Multiplexing with PSA™


PSA™ system has been designed to be compatible with other fluorescent markers, cell and tissue staining techniques, and other PSA™ imaging kits, enabling simultaneous visualization of multiple targets. The lower detection threshold of PSA™ compared to TSA and fluorescent secondary antibodies also allow the detection of two targets with primary antibodies raised in the same host species but without substantial crosstalk between the signals. PSA™ imaging is compatible with:
  • Fluorescent markers and counterstains (e.g., DAPI, Hoechst)
  • Fluorescent proteins (e.g., GFP, YFP, RFP)
  • Other Styramide™ reagents
  • Other PSA™ imaging kits
  • Tyramide reagents and tyramide imaging kits

iFluor™ 488 PSA™ Kit
iFluor™ 555 PSA™ Kit
Alexa Fluor® 594 tyramide
Alexa Fluor® 594 tryamide

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 (Cat No. 45205), followed by washing. Pan-Keratin was labeled with mouse anti-pan Keratin antibodies and iFluor™ 555 PSA™ Imaging Kit with goat anti-mouse IgG (Cat No. 45270). Nuclei were labeled with DAPI (Cat No. 17507). Images were acquired on a confocal microscope.

 

Flexible Workflow: compatible with IHC, ICC, ISH & flow cytometry


PSA™ imaging technology can be adapted to any application that supports the addition of HRP into its protocol and is compatible with sample types and fluorescence imaging platforms commonly used in immunological applications. When combined with conventional IHC, ICC, ISH, and FC applications, PSA™ imaging significantly increases detection sensitivity without losing image resolution or increased background noise.

ICC stain

Immunocytochemistry (ICC)

CD45 surface receptor stain of HL-60 cells. Hl-60 Cells were fixed with 4% formaldehyde, permeabilized, and labeled with 0.2 µg/mL anti-CD45 primary antibody. Cells were then stained with iFluor™ 647 Styramide™, and the fluorescence image was taken using the Cy5 filter set.


FISH stain

In Situ Hybridization (ISH)

Pan-centromeric staining via in situ hybridization using a biotinylated PNA probe. Jurkat cells were fixed and permeabilized using a standard protocol. The centromere of each chromosome was detected with streptavidin HRP conjugate and visualized with iFluor 488 Styramide reagent. Chromosomes were counterstained with DAPI.


Flow Data

Flow Cytometry

Flow cytometric analysis of pAKT in Jurkat cells using iF488 labeled goat anti-rabbit IgG method, tyramide method, or Styramide method. The Styramide amplification method provides a 10-fold-increase in signal over goat anti-rabbit IgG-iFluor™ 488 direct stain and 5-fold-increase over Alexa Fluor 488 tyramide stain.

  Goat anti-rabbit IgG-iFluor 488
  Alexa Fluor 488 Tyramide
  iFluor 488 Styramide
  Untained Control

 

PSA™ Imaging Workflow


PSA™ imaging exploits horseradish peroxidase (HRP) catalytic activity to generate high-density labeling of a target protein or nucleic acid sequence in situ. Like the conventional workflow of IHC, ICC, and ISH procedures, PSA™ imaging is easy-to-perform, comprising a few simple processes. In this workflow, the fluorescent secondary antibodies are replaced with poly-HRP secondary antibodies, and the only additional step is to incubate with labeled Styramide™.


Workflow for Power Styramide™ Signal Amplification (PSA™). With workflow similar to conventional ICC and IHC methods, PSA™ kits and Styramide™ reagents can achieve sensitive detection of desired targets in a few simple steps.
  1. Fix, permeabilize and block cells or tissue samples. Incubate sample with an unlabeled primary antibody, biotinylated primary antibody, or biotinylated nucleic acid probe.
  2. Add HRP-secondary antibody or HRP-streptavidin conjugate.
  3. Add iFluor™ dye Styramide™ working solution, allow for HRP-catalyzed deposition of Styramide™.
  4. Mount sample and detect Signal

 

Additional Resources



 

Table 1. Properties of Styramide™ reagents For Additional Resources

Styramide™
Mol. Wt.
Ex (nm)
Em (nm)
Filter Set
ε¹
Φ²
iFluor™ 350 Styramide™931.13345442DAPI20,0000.95
iFluor™ 488 Styramide™1037.99491514FITC75,0000.9
iFluor™ 546 Styramide™1326.69541557Cy3/TRITC100,0000.67
iFluor™ 555 Styramide™1023.15552567Cy3/TRITC100,0000.64
iFluor™ 568 Styramide™1243.60568587Cy3/TRITC100,0000.57
iFluor™ 594 Styramide™1341.71587603Cy3/TRITC180,0000.53
iFluor™ 647 Styramide™1231.63654669Cy5250,0000.25
iFluor™ 680 Styramide™1151.28683700Cy5220,0000.23
iFluor™ 700 Styramide™1158.44690713Cy7220,0000.23
iFluor™ 750 Styramide™1257.67759777Cy7275,0000.12
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).

Table 2. Styramide™ equivalents for common tyramides For Additional Resources

If you are using this tyramide reagent
Try this Styramide™ replacement
Price | Unit Size | Cat No.
Alexa Fluor® 350 tyramideiFluor™ 350 Styramide™$320 | 100 slides | 45000
Alexa Fluor® 488 tyramide, FITC tyramideiFluor™ 488 Styramide™$320 | 100 slides | 45020
Alexa Fluor® 546 tyramideiFluor™ 546 Styramide™$320 | 100 slides | 45025
Alexa Fluor® 555 tyramide, Cy3 tyramideiFluor™ 555 Styramide™$320 | 100 slides | 45027
Alexa Fluor® 568 tyramideiFluor™ 568 Styramide™$320 | 100 slides | 45030
Alexa Fluor® 594 tyramideiFluor™ 594 Styramide™$320 | 100 slides | 45035
Alexa Fluor® 647 tyramide, Cy5 tyramideiFluor™ 647 Styramide™$320 | 100 slides | 45045
Not Commercially AvailableiFluor™ 680 Styramide™$320 | 100 slides | 45050
Not Commercially AvailableiFluor™ 700 Styramide™$320 | 100 slides | 45055
Not Commercially AvailableiFluor™ 750 Styramide™$320 | 100 slides | 45065

Table 3. Available iFluor™ PSA™ Imaging Kits

iFluor™ PSA Imaging Kit
Secondary Antibody-HRP
Ex (nm)
Ex (nm)
Filter Set
Unit Size
Cat No.
iFluor™ 350 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP344448DAPI100 tests45250
iFluor™ 350 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP344448DAPI100 tests45200
iFluor™ 488 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP491514FITC100 tests45260
iFluor™ 488 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP491514FITC100 tests45205
iFluor™ 555 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP552567Cy3/TRITC100 tests45270
iFluor™ 555 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP552567Cy3/TRITC100 tests45220
iFluor™ 594 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP592619Cy3/TRITC100 tests45280
iFluor™ 594 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP592619Cy3/TRITC100 tests45230
iFluor™ 647 PSA™ Imaging KitGoat Anti-Mouse IgG-HRP649665Cy5100 tests45240
iFluor™ 647 PSA™ Imaging KitGoat Anti-Rabbit IgG-HRP649665Cy5100 tests45290

 

Product Ordering Information



 

Table 4. Ordering Info for PSA Products

Cat#
Product Name
Unit Size
Price
11060iFluor™ 488 tyramide1 mgDiscontinued
11061Azido-Cy5 tyramide1 mg310
11065Cy3 tyramide1 mg100
11066Cy5 tyramide1 mg100
11070AF488 tyramide reagent200 slides205
11075AF546 tyramide reagent200 slides205
11082AF594 tyramide reagent200 slides205
45000iFluor™ 350 Styramide *Superior Replacement for Alexa Fluor 350 tyramide*100 Slides310
45020iFluor™ 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide*100 Slides310
45025iFluor™ 546 Styramide *Superior Replacement for Alexa Fluor 546 tyramide*100 Slides310