Af2400

mouse anti-β-III-tubulin (Sigma #T8660; 1:2000), mouse anti-microtubule-associated protein 2a+b (MAP2, Sigma #M1406; 1:2000), rabbit anti-tyrosine hydroxylase (TH, Millipore #AB152; 1:600), mouse anti-synaptophysin (Sigma #S5768; 1:200), goat anti-forkhead box A2 (FOXA2 (Sigma #AF2400; 1:250), rabbit anti-GABA (Sigma #A2052; 1:2000), rabbit anti-GFAP (DAKO #Z0334; 1:4000), rabbit anti-TOM20 (Santa Cruz #SC-11415, 1:1000), goat anti-HSP60 (Santa Cruz Biotechnology #SC-1052, 1:200).

After removing the culture medium, cells were rinsed with PBS once and fixed with 4% paraformaldehyde (Wako, Japan) for 10 min at room temperature and blocked with 0.3% Triton-X-100 (Sigma-Aldrich, Germany) and 5% BSA in PBS for 30 min at room temperature. The fixed cells were then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: goat anti-PDX1 (1:100; R&D Systems, AF2419), mouse anti-NKX6.1 (1:50; DSHB, F55A10-s), goat anti-FOXA2 (1:100; R&D Systems, AF2400), mouse anti-HNF1B (1:100; Sigma-Aldrich, HPA002083), goat anti-HNF4A (1:100; Santa Cruz, sc-6556), rabbit anti-HNF6 (1:100; Santa Cruz, sc-13050), and mouse anti-NEUROD1 (1:100; Abcam, ab60704). After washing with PBS three times, cells were incubated for 60 to 120 min at room temperature with Alexa Fluor conjugated secondary antibodies (1:1000; Life Technologies). After washing with PBS twice, DAPI (1:1000; Life Technologies) with PBS was added to stain the nucleus. Images were captured using an Olympus microscope IX73.