C57BL/6 female mice (7 to 8 weeks old) were superovulated by intraperitoneally injecting with pregnant mares serum gonadotropin and human chorionic gonadotrophin and then mated to BDF1 male mice. Midday the following day was designated E0.5. The fertilized embryos (zygote) could be collected from oviducts. Then, E4.5 blastocyst embryos were obtained by culturing zygotes in G1 plus medium (no. 10128; Vitrolife, Göteborg, Sweden). E7.5 embryos were dissected from the pregnant mice, fixed for 30 min in 4% paraformaldehyde, and dehydrated in 30% sugar, and then, embryos were embedded in optimal cutting temperature compound (OCT), frozen, and sectioned for immunofluorescence.

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