Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.
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This work was supported by the National Institute of Biomedical Imaging and Bioengineering (R21 EB012188), the National Science Foundation (CBET-1351531), and the National Cancer Institute (R25CA153954). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; and Berron, Brad J., "Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells" (2014). Chemical and Materials Engineering Faculty Publications. 14.
S1 Fig. Quenching of fluorescent PBA by mounting medium.
journal.pone.0115630.s002.TIF (914 kB)
S2 Fig. Control study showing limited nonspecific labeling of cells.
journal.pone.0115630.s003.DOCX (13 kB)
S1 Table. Temporal staining intensity for Polymer Dye Labeling of nuclear pore complex.
journal.pone.0115630.s004.DOCX (17 kB)
S2 Table. Staining intensity for immunofluorescent labeling of nuclear pore complex.