Anti pdx1

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).

Cells were incubated with anti-Insulin (R&D Systems), anti-PDX-1 (R&D Systems) and anti-Nkx-6.1 (DSHB, USA) before being washed and analyzed with an LSRII Flow Cytometer (Becton Dickinson, USA).

For immunocytochemistry, hESCs were seeded on Matrigel-coated glass slides (SPL Life Sciences) and subjected to differentiation. The cells at day 8 (d8) were fixed in 4% (w/v) paraformaldehyde for 10–20 min at 4 °C and subsequently blocked for 20 min in PBS plus 0.2% Triton X-100 with 5% donkey serum (Dianova, Hamburg, Germany) and 1 mg/mL NaBH₄. Primary and secondary antibodies were diluted in PBS with 0.1% Triton X-100 and 0.1% donkey serum. Primary antibodies were incubated overnight at 4 °C. Secondary antibodies were diluted 1:500 and incubated for 1 h at room temperature. The primary antibodies anti-HNF1B (SantaCruz, sc-22840, Santa Cruz, CA, USA) and anti-PDX1 (R&D systems, AF2419, Minneapolis, MN, USA) were used. Secondary antibodies were obtained from Dianova (Hamburg, Germany) conjugated with AlexaFluor or Cy fluorophores. Finally, the slides were mounted with Immunoselect antifading mounting medium containing DAPI (Dianova). Stained cells were examined using an Olympus IX81 microscope (Olympus, Tokyo, Japan). For PDX1-positive cell counting, between 6–10 pictures for each condition from 3 experiments were taken and the cells were quantified using the plugin Image-based Tool for Counting Nuclei (ITCN) on Image J.