Fig. 3.

Multiplexed immunofluorescence of signal transduction pathways in CRC. 747 FFPE stage I–III CRC specimens arrayed on three TMAs were stained for 61 protein antigens including markers of epithelial, immune and stromal cell lineage, subcellular compartments, oncogenes, tumor suppressors, and significant posttranslational protein modifications. Pseudocolored images of signaling and regulatory molecule staining in one small field of view are shown. Nuclei are counterstained with DAPI and pseudocolored blue in all images. (A, 1) TMA core depicted in virtual H&E. (A, 2) Pseudocolored overlaid immunofluorescence of epithelial cells stained positive for pancytokeratin and stroma area stained positive for α-smooth muscle actin. (A, 3) Major subcellular compartments are detected by immunofluorescence staining of ribosomal protein S6 (cytoplasm) and Na⁺K+ATPase (plasma membrane), and the nucleic acid stain DAPI (nucleus). (B) Activation of mitogenic and anabolic signaling pathways in CRC cells. Multiplexed immunofluorescence of signaling protein expression and phosphorylation shows complex activation and repression patterns of regulatory and signal transduction pathways. The white arrow indicates a single cell expressing each feature and the cyan arrow indicates a cell with differential expression of features.