Rat anti e cadherin antibody

Total mRNA was isolated using TRIzol, reverse transcribed using Superscript III (Invitrogen), and analyzed using SYBR green PCR Master Mix (Applied Biosystems) coupled with 7900ht Fast RT-PCR system (Applied Biosystems). Human qRT-PCR primers were designed using Primer Blast (National Center for Biotechnology Information) and are described in Table S6. Proteins were extracted in PBS supplemented with 1% (vol/vol) Triton X-100, 1-mM EDTA, and protease inhibitor cocktail (Roche). E-cadherin protein was analyzed by Western blotting using rat anti–E-cadherin antibody (U3254; Sigma-Aldrich), anti–rat IgG-HRP secondary antibody (Jackson ImmunoResearch Laboratories, Inc.), and Western Lightning ECL kit (Thermo Fisher Scientific). E3.5 wild-type embryos were flushed in M2 medium (Sigma-Aldrich). Zona pellucida was removed with Tyrode’s acid (Sigma-Aldrich) and incubated for 10 min with anti–mouse antibody (Sigma-Aldrich) at 37°C and with guinea pig complement serum at 37°C for 30 min (Sigma-Aldrich). Three embryos or six resulting TE/ICMs were pooled together, and mRNA was isolated using TRIzol (Nishioka et al., 2009 (link)), reverse transcribed using Superscript VILO (Invitrogen), and analyzed using SYBR green PCR Master Mix. Mouse qRT-PCR primers (Table S7) were designed using Primer Blast.