SummaryImmunofluorescent staining is central to nearly all cell-based research, yet only a few fluorescent signal amplification approaches for cell staining exist, each with distinct limitations. Here, the authors present a novel, fluorescent polymerizationbased amplification (FPBA) method that is shown to enable similar signal intensities as the highly sensitive, enzyme-based tyramide signal amplification (TSA) approach. Being non-enzymatic, FPBA is not expected to suffer from nonspecific staining of endogenous enzymes, as occurs with enzyme-based approaches. FPBA employs probes labeled with photopolymerization initiators, which lead to the controlled formation of fluorescent polymer films only at targeted biorecognition sites. Nuclear pore complex proteins (NPCs; in membranes), vimentin (in filaments), and von Willebrand factor (in granules) were all successfully immunostained by FPBA. Also, FPBA was demonstrated to be capable of multicolor immunostaining of multiple antigens. To assess relative sensitivity, decreasing concentrations of anti-NPC antibody were used, indicating that both FPBA and TSA stained NPC down to a 1:100,000 dilution. Nonspecific, cytoplasmic signal resulting from NPC staining was found to be reduced up to 5.5-fold in FPBA as compared to TSA, demonstrating better signal localization with FPBA. FPBA's unique approach affords a combination of preferred attributes, including high sensitivity and specificity not otherwise available with current techniques. (J Histochem Cytochem 59:76-87, 2011)

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The growing need for medical diagnostics in resource limited settings is driving the development of simple, standalone immunoassay devices. A capillary flow device using polymerization based amplification is capable of blocking a microfluidic channel in response to target biomaterials, enabling multiple modes of detection that require little or no supplemental instrumentation.