Histological Analysis of Pancreatic Tumors

A cardiac terminal blood withdrawal was performed at the time of sacrifice. Pancreas and tumors were extracted from mice at 1, 2, 3 and 4 weeks after orthotopic or heterotopic tumor implantation. Tissues were formalin fixed for 48 h prior to paraffin embedding. Sections were cut at 5 µm thickness. Representative sections were stained with Hematoxylin and Eosin (H&E) and Sirius Red to detect fibrosis. IHC staining for CD31 (1:200; #77699, Cell Signaling Technology, Danvers, MA, USA), alpha smooth muscle actin (αSMA) (1:100; #A5228, Sigma-Aldrich, St Louis, MO, USA) and cleaved caspase-3 (1:200; #9664, Cell Signaling Technology) was performed according to standard protocols. Images were captured with a Nikon Eclipse microscope equipped with an Insight CMOS 5.1 digital camera. Whole slides were scanned using a NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics K. K, Hamamatsu City, Japan). Sirius Red staining was used to measure CPA for tumor and pancreas samples (Polasek et al., 2012 (link); Fuchs et al., 2013 (link)). All image analysis was performed using ImageJ (NIH).