Ab108422

Cells cultured in 2D were fixed with 4% paraformaldehyde (Santa Cruz, SC 281692, Dallas, TX, USA) blocked with a blocking solution comprised of 10% donkey serum and 0.1% Triton X-100 in PBS −/−. The cells were incubated with primary antibodies followed by secondary antibody incubation and 4′,6-diamidino-2-phenylindole (DAPI) staining. Immunofluorescence was observed using an Olympus IX73 microscope. The following primary antibodies were used to detect hPSC-associated markers: OCT4/POU5F1 (Abcam, ab19857, Cambridge, UK), NANOG (R&D systems, AF1997, Minneapolis, MN, USA), TRA-1-81 (ReproCELL USA, Inc., 09-001, Beltsville, MD, USA), TRA-1-60 (Millipore, MAB4360, Burlington, MA, USA) and SSEA-4 (Millipore, MAB4304, Burlington, MA, USA). The following primary antibodies were used to detect expression of germ-layer specific markers: SOX17 (R&D systems, AF1924, Minneapolis, MN, USA), FOXA2 (Abcam, Ab108422, Cambridge, UK), NESTIN (R&D systems, MAB1259, Minneapolis, MN, USA), PAX6 (Biolegend, #901301), α-actinin (Sigma, A7811, St. Louis, MO, USA) and SMA (Millipore, CBL171, Burlington, MA, USA).

Gastruloids were fixed over-night at 4°C in a 4% paraformaldehyde in PBS solution (Thermo Scientific, 15434389). Immune-labelling of gastruloids was performed as described previously (, Vianello et al., 2020b (link)). Primary antibodies used were as follows: rabbit anti-FOXA2 (Abcam, ab108422; 1:500), goat anti-SOX1 (R&D Systems, AF3369; 1:200), and mouse anti-cTnT (Invitrogen/Thermo Fisher Scientific, MA5-12960; 1:50). Nuclei were stained with 2 μg mL⁻¹ DAPI (Invitrogen/Thermo Fisher Scientific, D1306). Confocal Images were acquired on a Leica SP8 UP2 microscope. Images were processed using Fiji software (Schindelin et al., 2012 (link)). Fluorescent image intensity scales were adjusted equally.