Anti chromogranin a

Immunofluorescence and immunohistochemistry staining were performed as previously described [25] (link)–[28] (link). Non-specific binding was blocked and antibodies were diluted in PBS/Triton 0.1% solution containing 2% BSA (Sigma-Aldrich). The following antibodies were used at the indicated dilutions: anti-PCNA (1∶1000, Abcam), anti-lysozyme (1∶800, DakoCytomation), anti-UEA-1 (1∶1000, Sigma), anti-chromogranin A (1∶1000, Immunostar), anti-E-cadherin (1∶1000, BD), anti- β-catenin (1∶500, Cell Signaling), anti-IFABP (1∶500, kindly provided by Dr. Gordon [30] (link)) and FITC-conjugated anti-rabbit IgG (1∶200, Santa Cruz). TUNEL assay was performed according to the manufacturer’s protocol (Roche Diagnostic).

Cells were fixed in 4% paraformaldehyde solution (Affymetrix) for 20 min, then blocked and permeabilized in PBS solution containing 5% horse serum and 0.3% Triton for 1 h at room temperature. The cells were incubated with primary antibodies overnight at 4 °C followed by 1 h incubation with fluorescence-conjugated secondary antibodies (Alexafluor, ThermoFisher Scientific) at RT. For pSMAD2/3 staining, cells were permeabilized with ice-cold methanol at −20 °C for 10 min after fixation and prior to blocking. The following primary antibodies were used: anti-OCT4 (1:200, Santa Cruz), anti-SOX17 (1:500, R&D), anti-PDX1 (1:500, R&D), anti-SOX9 (1:1000, Millipore), anti-NKX6.1 (1:500, DSHB), anti-NKX2.2 (1:500, DSHB), anti-PAX6 (1:1000, Covance), anti-ISL1 (1:200, DSHB), anti-UCN3 (1:500, Pheonix Pharmaceuticals), anti-NGN3 (1:500, R&D), anti-chromogranin A (1:1000, Immunostar), anti-glucagon (1:2000, Sigma), anti-somatostatin (1:1000, DAKO), anti-ghrelin (1:500, Santa Cruz), anti-insulin (1:500, DAKO), and anti-cleaved caspase-3 (1:1000, BD Biosciences), anti-pSMAD2/3 (1:200, Cell Signaling).