Two-cell embryos were collected at day 2 of pregnancy and cultured in single-step medium for another 2-day period. Hatched blastocysts were added to the confluent monolayers of OPN-targeted siRNA or NC-siRNA pre-treated mESC in DMEM/F12 containing 10% cFBS for 48 h. Immunofluorescence was performed on this embryo-mESC co-culture as previously described [29] (link). Cells were incubated with a rabbit E-cadherin antibody (1∶100 dilution, Cell Signaling Technology, Boston, USA), goat polyclonal MMP-9 antibody (1∶100 dilution, Santa Cruz) or rabbit Ig G (1∶100 dilution, Santa Cruz), followed by the FITC conjugated secondary antibody (PIERCE, Rockford, IL, USA). Nuclei were stained with DAPI (Zhongshan Golden Bridge Bio-technology, Beijing, China). The trophoblast spreading area was quantified by measuring the E-cadherin signal area using the Image J software. A total of 10 embryos were calculated in each experiment, and the experiments were repeated three times.
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