Plan apochromat 20x 0.8 air objective

For whole-mount confocal analysis, stained embryos were mounted dorsal side down in PBTriton in Attofluor cell chambers (ThermoFisher A7816), using a small fragment of broken coverglass with small dabs of vacuum grease (Dow Corning) to mount the embryo on a #1.5 coverglass (Dow Corning). Embryos were then imaged by inverted confocal microscopy on either a Zeiss LSM700 equipped with a Plan-NeoFluar 40x/1.3 oil immersion objective, or a Leica SP8 equipped with a HC PL Apo 40x/1.3 oil immersion objective. Images were captured by tile-based acquisition of contiguous z-stacks of 50–150 μm depth with 0.9–1.2 μm optical slices and 0.3–0.5 μm z-steps. Tiled images were computationally stitched together with 10% overlap per tile using Zen (Zeiss) or LAS-X (Leica) software, resulting in visible seams in some images. Maximum-intensity projections of the entire z depth were created for analysis in the same software. For confocal imaging of cryosections, slides were imaged on an inverted Zeiss LSM700 equipped with a Plan-Apochromat 20x/0.8 air objective. Z-stacks of 10–14 μm depth were imaged with 1.8–2.0 μm optical slices and 1.0–1.2 μm z-steps. For bright-field imaging, embryos were imaged in PBTriton on a Zeiss Stemi 508 stereomicroscope equipped with a Canon EOS DSLR camera and EOS Utility software (Canon).

For immunofluorescence staining, undifferentiated and differentiated HUES9 parental and GFP-ABCG2 expressing HUES9 cells were seeded onto Matrigel coated (50 μg/mL) Imaging dishes (Zellkontakt). For labeling of pluripotency markers and the differentiation markers, we used the following primary antibodies: Oct4 (mouse, monoclonal, 1:50, SantaCruz,), cardiac Troponin-I (mouse monoclonal, 1:300, Sigma), beta-III Tubulin (mouse, monoclonal, 1:2000, RnD Systems), anti-human alpha Fetoprotein (AFP) (mouse, monoclonal, 1:500, Sigma), anti-alpha smooth muscle actin (SMA) (mouse, monoclonal, 1:100, Abcam), HNF4 (rabbit, polyclonal, 1:100, Abcam) and CK18 (mouse, monoclonal, 1:100, Abcam). As a primary antibody against GFP, anti-GFP (rabbit, polyclonal, 1:500, Abcam or chicken, polyclonal, 1:250, AVES) was used. 1 μM DAPI (Thermo Fisher Scientific) was used for nuclear staining. The stained samples were examined by a Zeiss LSM710 confocal laser scanning microscope, using Plan-Apochromat 20x (0.8) air objective (Zeiss) at 405, 488 and 633 nm excitations, respectively.