Figure 1

Outline of experimental approach for automated, quantitative multiplex immunofluorescence and biomarker measurements in defined regions of interest of prostatectomy tissue. A) Spectral profiles of each fluorophore in the spectral library used in the assay and profiles for tissue autofluorescence signals (AFL) and bright autofluorescence (BAFL) signals, respectively. B) A general outline of staining procedure for quantitative multiplex immunofluorescent biomarker measurements in tissue region of interest. SPP1 and SMAD4 were used as an example. Region of interest marker antibodies (KRT8 (CK8) and KRT18 (CK18) for total epithelium and KRT5 (CK5) and TRIM29 for basal epithelium) were directly conjugated to Alexa488 and Alexa 555, respectively. Biomarker antibodies were detected with a sequence of secondary and tertiary antibodies, as described. Colors in the table illustrate unique spectral positions of emission peaks for the indicated Alexa fluorophore dyes. C) A composite multispectral image (i) is unmixed into separate channels corresponding to AFL and BAFL, region of interest markers, and biomarkers, as indicated (ii). D) Definiens script-based tissue segmentation and biomarker quantitation. Moving through panels 1-6, from the composite image (1), first total epithelial regions are identified (2), followed by nuclear areas (3). The epithelial regions are further segmented into tumor shown in red, benign in green, and undetermined in yellow (4). Gray color denotes non-epithelial regions, e.g. stroma and vessels (4). Finally, biomarkers are quantified from tumor epithelium areas only, outlined in red (5 and 6).