Sulforhodamine B restaining as a whole-cell label allows visualizing one more fluorochrome and its application in assaying protein nucleocytoplasmic distribution.

Multiparametric image analysis is highly preferable in revealing complicated biological processes. The multiplexing capability is demanding and is limited by spectral overlap of fluorescent dyes and the number of fluorescent channels available in an imaging platform, especially when both nuclear and cytoplasmic staining are required for the analysis. Here, we introduce a retrospective method that enables cell labeling by restaining the cells with sulforhodamine B (SRB) using a positioning-stage equipped microscope and an automated image registration technique. Using signal transducers and activator of transcription 1 (STAT1) nuclear translocation as an example, this method was shown to be able to improve the reliability and multiplexing capability of protein distribution assays. The application of this method in a three-fluorescent channel platform is functionally equal to a conventional four-color assay and enables the correlation between cellular distributions of two proteins expressed at very low levels to be detected. We have demonstrated this application using STAT3 and syndecan-1. For the first time, we found that the two proteins were correlated at both the nuclear:cytoplasmic distribution and expression levels. This experimental direction could advance our understanding as to how these molecules function. The simplicity and automation of this method makes it easily applicable into other imaging assays. Other dyes can be used in a similar way as substitutes of SRB.