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.
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.
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.
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
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.
In Situ Hybridization (ISH)
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. 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 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
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.
Fix, permeabilize and block cells or tissue samples. Incubate sample with an unlabeled primary antibody, biotinylated primary antibody, or biotinylated nucleic acid probe.
Add HRP-secondary antibody or HRP-streptavidin conjugate.
Add iFluor® dye Styramide™ working solution, allow for HRP-catalyzed deposition of Styramide™.
Mount sample and detect Signal
Table 1. Properties of Styramide™ reagents For Additional Resources