Tissue samples of human cholangiocarcinomas and corresponding samples of normal liver tissue (n=18) were obtained with Institutional Review Board approval. Liver tissue from euthanized mice was fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned into 3.5 μm slices. Human CCA specimens were prepared in a similar manner. Paraformaldehyde-fixed, paraffinembedded liver tissue sections were deparaffinized, hydrated and incubated with primary antibody overnight at 4°C. Antibody sources and dilutions are as follows: pAKT (1:50), YAP (1:25), and phospho-STAT3 (1:200) from Cell Signaling (Danvers, MA); α-smooth muscle actin (1:500), ST2 (1:500), and CK-19 (1:500) from Abcam (Cambridge, MA); PanCK (1:500; Dako, Carpinteria, CA), SOX9 (1:1000; Millipore, Billerica, MA), HepPar1 (1:40; Thermo Fisher Scientific, Waltham, MA). PanCK antibody was used as it only recognizes biliary epithelia in mice liver.19 (link) Bound antibodies were detected with biotin conjugated secondary antibodies and diaminobenzidine (Vector Laboratories, Burlingame, CA) as a substrate and the tissue slices were counterstained with hematoxylin.