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

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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

Superior Detection Sensitivity

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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.

Multiplexing with PSA™

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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

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

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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.

PSA™ Imaging Workflow

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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™.

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

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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).

Product Ordering Information

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