Power Styramide™ Signal Amplification (PSA™)

iFluor™ 488 Styramide

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 a much higher reactivity than tyramide radicals, making imaging with PSA™ significantly faster, more robust and sensitive than conventional TSA labeling. And, since the added labeled-Styramide™ are deposited in close proximity of the HRP-target site, there is minimal diffusion-related loss of resolution. PSA™ imaging technology can be readily added to any application that allows for the integration of HRP into it’s 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 a more robust and expeditious labeling of Styramide™ at the HRP-target interaction site resulting in a 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 significant reduction in primary antibody
• PSA™ imaging kits are easy-to-use and provide sufficient reagents for 100 tests

iFluor™ 594 Styramide™
(Cat No. 45035)

Alexa Fluor® 594 tyramide
(Cat No. 11082)

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 as well as overcome insufficient immunolabeling caused by poor fixation procedures or low-levels of target expression.

PSA™ system has been designed to be compatible with other fluorescent makers, cell and tissue staining techniques, and with other PSA™ imaging kits, enabling simultaneous visualization of multiple targets. The lower detection threshold of PSA™ compared to TSA and fluorescent secondary antibodies also allows 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, Hoechsts)
• Fluorescent proteins (e.g. GFP, YFP, RFP)
• Other Styramide™ reagents
• Other PSA™ imaging kits
• Tyramide reagents and tyramide imaging kits

iFluor™ 488 PSA™ Kit
(Cat No. 45205)

DAPI
(Cat No. 17507)

iFluor™ 555 PSA™ Kit
(Cat No. 45270)

Merged

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. Centromere of each chromosome was detected with streptavidin HRP conjugate and visualized with iFluor 488 Styramide reagent. Chromosomes were counterstained with DAPI.

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.

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.

PSA™ imaging exploits the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ. Similar to the conventional workflow of IHC, ICC and ISH procedures, PSA™ imaging is easy-to-perform comprising of 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 sample. Incubate sample with 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

Table 1. Properties of Styramide™ reagents For Additional Resources

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

Table 3. Available iFluor™ PSA™ Imaging Kits

Table 4. Ordering Info for PSA Products