Anti gata6

Wild-type CD1 and Nanog^+/+, Nanog^+/- and Nanog^-/- embryos were generated by in-house breeding and natural mating. Detection of copulation plug confirmed successful mating; the resulting embryos were then considered embryonic day (E) 0.5. Embryos were isolated in M2 medium (Millipore). Embryos were prepared for immunofluorescence as previously described (89). Primary antibodies used were: anti-NANOG (eBiosciences 14–5761, 1:100), anti-GATA6 (R&D, AF1700, 1:200). Nuclei were stained using DAPI or Hoechst (1:1000, Invitrogen). Embryos were mounted on microscopy slides with Vaseline bridges to prevent their crushing. Three independent immunofluorescence stainings, each with E3.5 and E4.5 embryos from 7 litters, were performed for the first wild-type data set.
Nanog mutant embryos were obtained as previously described [4 (link)] and genotyped by NANOG antibody staining.

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).

Whole-cell protein extracts were isolated from EPS-1, EPS-2, EPS-Gata6KO-1, and EPS-Gata6KO-2 cell line using RIPA lysis buffer (Beyotime, P0013B) supplemented with protease inhibitor cocktail (Roche, 04693132001). Protein extracts were loaded onto SDS-polyacrylamide gel. Gel was run at 80 V for 30 min and 120 V for 1.5 h. Proteins were transferred for 2 h at 200 mA to a PVDF membrane (IPVH00010, Millipore). Blots were incubated in rapid blocking buffer (Epizyme Biomedical Technology, PS108) for 30 min. Primary antibodies were incubated overnight at 4°C, and secondary antibodies were incubated at RT for 1 h. The signals were measured using ECL reagents (GE) and visualized by the ChemiDoc MP imaging system (Bio-Rad).
The primary antibodies and dilutions used were as follows: anti-GATA6 (1:400; R&D Systems, AF1700), anti-β-Actin (1:2,000; Proteintech, HRP-66009). The primary antibodies and dilutions used were as follows: anti-goat IgG (H+L) (1:5,000; Beyotime, A0181), anti-mouse IgG (H+L) (1:5,000; Beyotime, A0216).

Cells were fixed [15 (link)] and stained with unconjugated monoclonal anti-α-smooth muscle actin (α-SMA; Sigma-Aldrich; cat. number: A5228) or anti-GATA6 (R&D Systems Minneapolis, USA; cat. number: AF1700) primary antibody or the matched isotype control for 1 hour at room temperature in the dilution according to manufacturer's instructions. AlexaFluor488 (α-SMA) or AlexaFluor568 (GATA6) secondary antibodies were applied for fluorescent detection, and nuclei were counterstained with DAPI. The fluorescence membrane stain DiSC₃(5) was used in final concentration of 5 μM.