Guinea pig complement

To isolate the inner cell mass (ICM) of E4.0 embryos, embryos were incubated in pre-warmed rabbit anti-mouse serum (Rockland Immunochemicals, #110–4101) at a 1:5 dilution in M2 medium for 1 hr in a humid chamber at 37°C. After briefly rinsing in M2 medium, embryos were incubated in pre-warmed guinea pig complement (Sigma, cat No. S1639) at a 1:5 dilution in M2 medium for 45 min to 1 hr in a humid chamber at 37°C. The embryos were then repeatedly pipetted using a finely-pulled glass Pasteur pipette to remove trophectoderm cells. Isolated ICMs were treated with 0.05% trypsin for 10 min for mild dissociation, then incubated in M2 media with 10% fetal bovine serum (FBS) for 10 min to neutralize the trypsin. ICMs were then rinsed in 1X PBS and permeabilized through sequential transfers into ice-cold CSK for 1 min, ice-cold CSK containing 0.4% Triton X-100 buffer for 5 min, followed twice with ice-cold CSK for 1 min each. ICMs were mounted on a glass coverslip coated with 1X Denhardt’s solution in a small drop of ice-cold solution of 1X PBS containing 1% paraformaldehyde and 20% CSK buffer. Excess solution was aspirated off and the coverslip air-dried for 15 min. The ICMs were then fixed in cold 3% paraformaldehyde for 10 min. After fixation, the coverslips were rinsed 3X in 70% ethanol and stored in 70% ethanol at −20°C prior to use.