Ab5535

Cultures were fixed in 3.7% paraformaldehyde (Fisher Scientific) for 10 min, permeabilized in 0.2% Triton X-100 (MilliporeSigma, X100–100 ml) for 10 min, and incubated overnight at 4°C with primary antibodies diluted in a blocking buffer containing 1% bovine serum albumin (Prometheus Protein Biology Products, 25–529) and 0.1% Tween-20 (MilliporeSigma, P7949-100 ml). The cultures were subsequently washed twice with the blocking buffer and incubated for 2 h with secondary antibodies. DAPI (1:1,000, ThermoFisher Scientific) was added for 30 min before washing twice in DPBS. Antibodies used include: SOX9 (1:1,000, MilliporeSigma, AB5535), SOX10 (1:100, R&D Systems, AF2864), TUJ1 (1:500, MilliporeSigma, MAB1637), PRPH (1:1000, Novus Biologicals, NB300137), Alexa Fluor 488 donkey anti-mouse (1:1,000, Invitrogen A-21202), Alexa Fluor 555 donkey anti-goat (1:1,000, Invitrogen, A-21432), Alexa Fluor 647 donkey anti-rabbit (1:1,000, Invitrogen, A-31573). Negative controls included unstained cultures and positively stained cultures known not to express the antigens of interest. Stained cultures were imaged using a Leica DMI6000B microscope and a DFC365FX camera.

Transfected embryos were collected at HH10 and fixed in 4% PFA. Subsequently, the embryos were washed in TBST (1X TBS with 0.1% TritonX-100) and TBTD (1X TBST with 1% DMSO). After blocking with 10% Donkey Serum (Equitech-Bio, sd300500) in TBTD for 1 hour, embryos were incubated with primary antibodies (SOX9: Millipore Sigma ab5535 1:200; SOX2: R&D Systems AF2018 1:200) at 4°C overnight. The following day, embryos were washed in TBTD 3 times and incubated with secondary antibodies (Alexa Fluor 488 and 633: Thermo Fisher Scientific, A21206 and A21126, respectively) and DAPI. Whole embryos were imaged using a Zeiss Imager.Z2 fluorescent microscope. For cryosectioning, embryos were transferred to 5% sucrose (Sigma S7903) in 1X PBS for 2 hours at room temperature, followed by 15% sucrose in 1X PBS at 4°C overnight. Embryos were then incubated at 37°C in 7.5% gelatin (Sigma G1890) in 15% sucrose PBS solution for 2 hours and embedded in silicone molds underwent flash frozen in liquid nitrogen. The samples were sectioned using a CryoStar cryotome. The sections were transferred onto charged glass slides at room temperature overnight. The next day, the slides were incubated in 1X PBS at 42°C for 15 min and 1X PBS at room temperature for 10 min 3 times to wash off the gelatin. After mounting with Fluoromount, slides were imaged using a Zeiss Imager.Z2 fluorescent microscope.

Liver organoids were extracted from Matrigel (with Cell Recovery Solution, Corning) or PEG gels (with 1 mg ml− 1 Dispase (Gibco) for 10 min at 37 °C) and fixed with 4% paraformaldehyde (PFA) in PBS (20 min, room temperature). Organoids in suspension were centrifuged (1000 r.p.m., 5 min) to remove the PFA, washed with ultrapure water and pelleted. The organoids were then spread on glass slides and allowed to attach by drying. Attached organoids were rehydrated with PBS and permeabilized with 0.2% Triton X-100 in PBS (1 h, room temperature) and blocked (1% BSA in PBS) for 1 h. Samples were then incubated overnight with phalloidin-Alexa 488 (Invitrogen) and primary antibodies against Epcam (1:50, eBioscience, G8.8), Krt19 (1:100, Abcam, ab15463), E-cadherin (1:100, Cell Signaling, 24E10), Sox9 (1:50, Millipore, AB5535), Albumin (1:50, R&D systems, MAB1455), Hnf4α (1:50, Santa Cruz, C19), ZO-1 (1:50, Invitrogen, 61-7300); YAP (1:50, Cell Signaling, 4912 S). Samples were washed with PBS and incubated for 3 h with secondary antibodies Alexa 488 donkey-α-rabbit, Alexa 568 donkey-α-mouse, Alexa 647 donkey-α-goat (1:1000 in blocking solution; Invitrogen). Following extensive washing, stained organoids were imaged by confocal (Zeiss LSM 710) mode. Dapi was used to stain nuclei.