A0231

Whole cell lysates were prepared with lysis buffer (20 mM Tris HCl pH 7.5, 137 mM NaCl, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1% Triton X-100, 10% glycerol and 1.5mM MgCl₂). The following primary antibodies were used: anti-MSX2 (HPA005652, Sigma-Aldrich, 1:800), anti-human Chorionic Gonadotropin (hCG) (A0231, Dako, 1:1,000), anti-ENDOU (HAP067448, Sigma-Aldrich, 1:1,000), anti-tubulin (ab6160, Abcam, 1:2,000), anti-FlagM2 (F1804, Sigma-Aldrich, 1:2,000), anti-GCM1 (HPA011343, Sigma-Aldrich, 1:1,000).

Placental tissue (seventh week of gestation) was embedded in paraffin and processed as described (45 (link)). Utilization of tissues and all experimental procedures were approved by the Medical University of Vienna ethics boards (No. 084/2009) and required written informed consent. Cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) for 20 min at 4 °C, permeabilized, and blocked for 30 min in 4% donkey serum and 0.1% Triton X-100 in PBS. The following primary antibodies with given dilutions were used: anti-MSX2 (HPA005652, Sigma-Aldrich, 1:250), hCG (A0231, Dako, 1:300), anti-ENDOU (HAP067448, Sigma-Aldrich, 1:250), and anti-ZO-1 (ab216880, Abcam, 1:1,000). Alexa Fluor–conjugated secondary antibodies (A-21206, A-21207, Invitrogen) were applied at 1:1,000 in 4% donkey serum and 0.1% Tween-20 in PBS blocking solution. Cells were counterstained with DAPI and imaged using Zeiss Imager A2 microscope with Zen 2012 software.