Pre-implantation blastocyst embryos at stages up to E3.5 (E4.0 for hybrid embryos) were recovered by flushing the uterus with M2 medium (Sigma). Embryos at E3.75 (E4.25 for hybrid embryos) and later were dissected out from the uterus. The embryos were staged on the basis of their morphology and number of cells per ICM.
When applicable, the zona pellucida was removed using acidic Tyrode’s solution (Sigma), and embryos were washed twice with M2 medium (Sigma). ICM was then isolated from all stage blastocysts by immunosurgery. Briefly, blastocysts without zona pellucida were quickly cultured in anti-mouse red blood cell serum from rabbit (Rockland) for 30 min then in guinea pig complement serum (Sigma) for 15–30 min. Only TE cells were killed and debris carefully removed with a glass pipette48 (link).
When applicable, the zona pellucida was removed using acidic Tyrode’s solution (Sigma), and embryos were washed twice with M2 medium (Sigma). ICM was then isolated from all stage blastocysts by immunosurgery. Briefly, blastocysts without zona pellucida were quickly cultured in anti-mouse red blood cell serum from rabbit (Rockland) for 30 min then in guinea pig complement serum (Sigma) for 15–30 min. Only TE cells were killed and debris carefully removed with a glass pipette48 (link).