Power Styramide™ Signal Amplification a Superior Alternative to Tyramide Signal Amplification

Ultra-Sensitive Detection: High-Resolution Imaging of Low-Abundant Targets

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Targets of functional importance such as transcription factors, integral membrane proteins and cell-surface cytokine receptors have endogenous expression levels far below the detection capabilities of labeled antibodies. Not only does the abundance of these targets vary by several orders of magnitude, but their spatiotemporal organization within the cell is dynamic making it challenging to visualize. AAT Bioquest's novel Power Styramide™ Signal Amplification (PSA™) system provides an ultra-sensitive method for detecting low-abundance targets in immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (ISH) applications. PSA™ imaging technology combines the superior brightness and photostability of iFluor^® dyes with HRP"mediated Styramide™ signal amplification to generate fluorscence signals with significantly higher precision and sensitvity, more than 100 times greater than standard ICC/IHC/ISH methods and up to 50 times greater than traditional tyramide signal amplification techniques (Figure 1).



Similar to tyramide signal amplification, PSA™ labeling utilizes an enzyme-mediated detection method that harnesses the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of iFluor^®dye-Styramide™ conjugates onto a target protein or nucleic acid sequence in situ. In the first step of this process, a probe, such as a primary antibody or nucleic acid sequence, binds to the target via immunoaffinity or hybridization, respectively. This initial complex is then detected with an HRP-labeled secondary antibody or HRP-streptavidin conjugate (if a biotinylated primary antibody was used in the initial detection of the target). Next, multiple copies of a labeled Styramide™, such as iFluor^® 488 Styramide™ or iFluor^® 555 Styramide™, are activated by enzymatic reaction with HRP. Lastly, the highly reactive, short-lived Styramide™ radicals covalently couple with tyrosine residues proximal to the HRP"target interaction site, resulting in minimal diffusion-related loss of signal localization (Figure 2). Any unbound labeled Styramide™ radicals quickly dimerize, and are washed away to eliminate background.

PSA™ Workflow

 

PSA™ Imaging - Greater Sensitivity for Low-Abundance Targets

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The Styramide™ signal amplification technique used in our PSA™ Imaging kits utilizes the catalytic activity of HRP for the covalent deposition of labeled Styramide™ proximal to the target of interest. The enhanced sensitivity of the PSA™ Imaging kits over conventional immunofluorescence methods using fluorescently labeled antibodies or other tyramide signal amplification technologies is in part due to improvements in the reagents for each amplification step. Figure 1 shows an example of the improved sensitivity of Styramide™ signal amplification to detect EpCam in human lung adenocarcinoma in comparison to conventional tyramide labeling.

The PSA™ Imaging kits enhance the specific fluorescence signal by utilizing iFluor^® dye labeled Styramide™ conjugates, which react with HRP to covalently deposit brightly fluorescent and photostable iFluor^® dye Styramides™ on tyrosine residues and other similar molecules in close proximity. Furthermore, Styramide™ radicals have a significantly higher reactivity than traditional tyramide radicals making PSA™ labeling abundantly faster, more robust and sensitive than traditional tyramide signal amplification systems (Figure 3).



PSA™ Imaging kits also employ several strategies to maximize convenience and performance. First, Styramide™ conjugates allow for significantly less consumption of primary antibody compared to coventional immunofluorescence techniques to acheive sensitive detection of low-abundance target molecules. Even with much less primary antibody, PSA™ technology provide detection sensitivities similar to or greater than those obtained using a fluorescently labeled secondary antibody or tyramide signal amplification in an ICC application (Figure 3). Using fewer antibodies per experiment will save on the cost of primary antibodies, a major expense in ICC and IHC workflows. Also, more experiments can be accomplished using a single vial of primary antibody. Given that some primary antibodies show significant lot-to-lot variation, using a single lot throughout a project can produce more reliable results that are consistent from experiment to experiment. Second, these kits contain highly cross-adsorbed secondary antibodies to help ensure specificity for the target primary antibody with minimal cross-reactivity with other antibody species. For example, HRP"conjugated goat anti"mouse IgG exhibits no detectable reactivity to mouse proteins or IgG derived from other species such as goat, bovine, human, or rat. Lastly, PSA™ Imaging kits include a robust labeling protocol with all the necessary reagents to perform 100 tests.

PSA™ amplification is multiplexable

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The PSA™ Imaging kits are available with five spectrally distinct iFluor^® dye Styramide™ conjugates and two different HRP secondary reagents (Table 1). Additionally, there are eleven stand-alone iFluor^® dye Styramide™ conjugates spanning the UV to NIR for further flexibility in designing multi-parametric applications (Table 2). This range of choices allows high-resolution detection and visualization of multiple signals in a single cell or tissue sample. In addition to multiplex detection using primary antibodies from different species (Figures 4), PSA™ imaging is also compatible with experiments using GFP and RFP fusions as reporters of gene expression.