Nuance system

The quantitative analysis of protein expression was carried out using the Nuance system (Perkin-Elmer, Waltham, MA). Multispectral 8-bit image cubes of relevant areas on each slide were acquired in brightfield mode at 20nm intervals from 420 to 720nm, using a 20× objective lens and 1×1 binning. A spectral library containing the characteristic wavelength emission curves of MB and DAB was created by sampling pure spectra from slides stained with single dyes, i.e. MB only and DAB only. Blue and red pseudo-colors were assigned to visualize MB and DAB, respectively. The image cubes, initially shown as pseudo-color composites of both blue and red, were unmixed into individual components by the Nuance 3.0.0 software version. For quantitation of protein expression, 100–300 cells per slide were manually designated as regions of interest (ROIs). Using the spectral library as reference, the system measured the amount of target signal (intensity of red pseudo-color) within each ROI, automatically converting spectral data into optical density (OD) units. Final data are displayed as average OD/cell. All histograms shown in Figures 3–7 are based on the quantitative assessment of protein expression by MIA. Exemplary MIA-generated high-power images of protein expression and quantitative analysis are shown in Figure 2.