For the detection of pluripotency and differentiation markers, cells grown in 24-well plates were fixed with 4% paraformaldehyde for 20 minutes. Cells were permeabilized and blocked with 10% donkey serum and 0.1% Triton X-100 in PBS. Subsequently, cells were stained with primary antibodies overnight at 4°C and finally incubated with fluorochrome-labeled secondary antibodies (Invitrogen, UK). The primary antibodies used for detecting pluripotency markers were: anti-OCT4 (SC-5279, Santa Cruz Biotech, USA), anti-SOX2 (AF2018, R&D, UK), anti-NANOG (AF1997, R&D, UK). The primary antibodies used for detecting endoderm markers were: anti-SOX17 (AF1924, R&D, UK), anti-CXCR4 (MAB173-100, R&D, UK and anti-GATA4 (SC-25310, Santa Cruz Biotech, USA). The primary antibodies used for detecting mesoderm markers were: anti-Brachyury (AF2085, R&D, UK), anti-EOMES (Ab23345, Abcam, UK) and anti-MIXL1 (SC-98664, Santa Cruz Biotech, USA). The primary antibodies used for detecting neuroectoderm markers were: anti-NESTIN (AB22035, Abcam, UK), and anti-SOX1 (AF3369, R&D, UK) anti-SOX2 (AF2018, R&D, UK). The secondary antibodies used were: Donkey anti-goat AF488 (Invitrogen, UK), Donkey anti-mouse AF488 (Invitrogen, UK), Donkey anti-rabbit AF488 (Invitrogen, UK). Additionally, DAPI staining was used to label cell nucleus in order to facilitate cell segmentation.
