Deidentified, formalin-fixed, paraffin-embedded tissue was procured from four cases of prostate cancer and 10 cases of PDAC through the Cooperative Human Tissue Network with approval from the Vanderbilt Institutional Review Board. 5-μm sections were processed as described (Shi et al., 2014 (link)) and labeled with antibodies to Fn for colorimetric analysis. Detection was performed using Vectastain Elite ABC kit (Vector Laboratories) after a reaction with 3,3′-diaminobenzidine (Vector Laboratories). For fluorescent analysis, antibodies to smooth muscle α-actin (αSMA) were detected using the Vectastain Elite ABC kit, followed by tyramide signal amplification with the TSA Plus Cyanine 3 kit (PerkinElmer), followed by heat inactivation. Binding of anti–Fn antibodies was detected with Cy2–anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, Inc.). Slides were counterstained with hematoxylin (Sigma Aldrich) or Toto3 (Thermo Fisher Scientific) and DAPI (Sigma-Aldrich). Colorimetric images were obtained on an Axioskop 40 microscope (ZEISS), and fluorescent images were captured on the Quorum WaveFX spinning disk confocal system with Nikon Eclipse Ti microscope as described in Microscopy.