Anti mouse rabbit serum

Embryo and cell manipulations were carried out under a dissecting microscope (Leica Microsystems). The zona pellucida was removed using acid Tyrode’s solution (Sigma) at E4.0. E4.5 blastocysts were subjected to immunosurgery as previously described (Solter and Knowles, 1975 (link)). In brief, blastocysts were incubated for 45-60 minutes in a 1:5 dilution of anti-mouse rabbit serum (Sigma) in N2B27, washed in N2B27 and further incubated for 30-60 minutes in a 1:5 dilution of rat serum (in-house) in N2B27 for the complement reaction. The ICM was subsequently cleaned from residual trophectoderm with a narrowly fitting glass pipette. Isolated ICMs were culture in N2B27 at 37°C and 5% CO₂ with or without 1 μg/ml Dox an equivalent to E5.5 in which PrE lineage cells surround the matured EPI.

Embryo and cell manipulations were carried out under a dissecting microscope (Leica Microsystems). The zona pellucida was removed using acid Tyrode’s solution (Sigma). Blastocysts from E3.5-E4.5 were subjected to immunosurgery as previously described (Solter and Knowles, 1975 (link)). In brief, blastocysts were incubated for 45-60 minutes in a 1:5 dilution of anti-mouse rabbit serum (Sigma) in N2B27, washed in N2B27 and further incubated for 30-60 minutes in a 1:5 dilution of rat serum (in-house) in N2B27 for the complement reaction. The ICM was subsequently cleaned from residual trophectoderm with a narrowly fitting glass pipette. Single-cell dissociation of ICMs was performed in a 1:1 mixture of Accutase and 0.025% trypsin (Invitrogen) plus 1% chick serum (Sigma). Cells were dissociated by repetitive using blunted microcapillaries (Global Scientific or Harvard apparatus) and washed in Blast (Origio) or N2B27.