Journal
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
Volume 59, Issue 1, Pages 76-87Publisher
SAGE PUBLICATIONS LTD
DOI: 10.1369/jhc.2010.955948
Keywords
immunofluorescent cell staining; immunocytochemistry; signal amplification; polymerization; non-enzymatic
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Funding
- NIH [R21 CA 127884]
- National Science Foundation
- state of Colorado
- University of Colorado Technology Transfer Office
- NATIONAL CANCER INSTITUTE [R21CA127884] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING [R21EB012188] Funding Source: NIH RePORTER
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Immunofluorescent 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 polymerization-based 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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