Albumin

Troponin I (Santa Cruz Biotechnology), MLC2v (Proteintech Europe), Tuj1 (Santa-Cruz), FOXA2 (Santa Cruz), Alpha foeto protein (Sigma-Aldrich), Albumin (Cedarlane).

Cultured cells were fixed with 4% PFAfor30 minutes and made permeable using 0.5% Triton X-100 in PBS for 15 minutes. Cells were treated with 3% BSA in PBS for 30 minutes followed by overnight incubation with primary antibody at 4°C. Antibodies used were HNF4A (Santa Cruz, #sc-1556, 1:250), Albumin (Cedarlane, #CL2513A, 1:500), and AFP (Sigma, #A8452, 1:250). Cells were rinsed with PBS 3 × 5 minutes and incubated with DAPI (1 μg/ml) and secondary antibody for 1 hour at room temperature. Alexa fluor antibodies (594 nm anti-goat, 488 nm anti-rabbit, 488 nm anti-goat) were used at 1:1000 dilution. Images were processed using Adobe Photoshop to optimize brightness/contrast. Control and experiment wells were processed identically.

Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 minutes, followed by permeabilization-blocking with 5% Donkey Serum and 0.1% Triton X-100 in PBS for 1 hour. Cells were then stained with primary antibodies overnight at 4 °C. Primary antibodies used are as follows; OCT4 (Santa Cruz, Sc9801, 1:200), Brachyury (Cell Signaling Technology, 81694, 1:1000), SOX17 (Cell Signaling Technology, 81778, 1:1000), PAX6 (Biolegend, 901301, 1:200), TNNI3 (R&D Systems, MAB8594, 1:100), NKX2.5 (Sigma, SAB1408911, 15 μg/ml), Flavivirus Group Antigen (Millipore Sigma, MAB10216, 1:200), GABA (Sigma, A2052, 1:1000), vGLUT1 (Sigma, AMAb91041, 1:5000), CUX2 (Abcam, ab130395, 1:500), TUBB3/TUJ1 (Biolegend, 801201, 1:1000), CTIP2 (Novus, NBP2–61702, 1:250), MAP2 (Thermo Fisher Scientific, PA5–17646, 1:500), HNF4a (Santa Cruz, sc-6556, 1:250), AFP (Sigma, A8452, 1:1000), FOXA2 (BD, 561580, 1:200), Albumin (Cedarlane, CL2513A, 1:250) and DAPI (1:1000). Secondary antibodies used are as follows; Donkey anti-Rabbit 488 (Thermo Fisher Scientific, A21206, 1:2000), Donkey anti-Mouse 568 (Thermo Fisher Scientific, A10037, 1:2000), Donkey anti-mouse 488 (Jackson ImmunoResearch, 715-545-150, 1:500), Donkey anti-rabbit 594 (Jackson ImmunoResearch, 711-585-152, 1:500). Fluorescence images were taken with the Leica DMi8 microscope.