Abstract
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.
Highlights
The determination of spatial patterns of protein expression in biological samples is a cornerstone of modern clinical diagnostic and biological research
Our goal is to develop a colorimetric alternative to enzymatic amplification which is not hampered by non-specific amplification by endogenous enzymes or through diffusional loss of signal localization
Our approach, ‘‘Polymer Dye Labeling,’’ is a multi-step process where 1) polymerization initiator is localized to the site of antigen expression, 2) an interfacial polymer coating is grown from the surfacegrafted initiator, and 3) dye is loaded into the polymer
Summary
The determination of spatial patterns of protein expression in biological samples is a cornerstone of modern clinical diagnostic and biological research. While direct labeling of the target antibody is sufficient for localization of abundant proteins in fluorescent imaging, amplification of the signal is typically required to label proteins for brightfield observation of samples where dilute proteins can be difficult to observe colorimetrically. Nonspecific HRP signal is common from endogenous peroxidases naturally residing in the tissue [2]. The presence of these active enzymes in the sample tissue requires additional sample processing to quench their activity [3]. Fine localization of HRP staining is an empirical process, where over-amplification commonly results in significant diffusion of the signal away from the targeted protein expression
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