Cells for immunocytochemistry were grown on ibidi µ-slides and stained as described in Kalmar et al. (2009 (link)). Primary antibodies were anti-NANOG (1:200; eBiosciences, 14-5761), anti-FLAG (1:1000; Sigma M2, F3165), anti-GATA6 (1:200; R&D, AF1700) and anti-GATA4 (1:200; Santa Cruz, sc-9053). Detection was performed using Alexa Fluor-conjugated secondary antibodies at 4 μg/ml (Molecular Probes). Nuclei were visualized using Hoechst 33342 dye at 100 μg/ml (Molecular Probes, H1399). Cells were imaged on a Zeiss LSM700 confocal microscope with a 40× oil immersion lens (NA 1.3).
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