Figure 2

Tissue annotation, quality control procedures and assay reproducibility. A) Tissue annotation and quality control procedures. Left: A representative H&E-stained section of a human prostatectomy sample showing four (blue) and two (green) 1 mm diameter circles placed over the regions with the highest and lowest Gleason pattern, respectively, as annotated by an expert pathologist. Two cores (1 mm diameter each) were taken from two of the four blue regions to generate TMA blocks. Right: A consecutive section of the same prostatectomy sample was stained with DAPI and KRT8/KRT18-Alexa488. Areas with bright staining of prostate epithelium by KRT8/18 cytokeratin antibodies were considered good quality regions, while areas with little or no staining (as indicated within the yellow punctate area) were considered of low quality and not deemed suitable for TMA construction. B) Intra-experimental reproducibility. Two consecutive sections from a prostate tumor test TMA were stained in the same experiment. Images were acquired using the Vectra system and processed with a Definiens script. Scatter plots compare mean values of KRT8/18, PTEN, and SMAD4 staining intensities from the same cores of the consecutive TMA sections. Linear regression curves, equations, and R² values were generated using Excel software.