Guinea pig complement serum

At 120 hours of embryo culture growth, differential staining was performed on all expanding, hatching, and hatched blastocysts, using the protocol described by Pampfer et al. [11 (link)]. Briefly, the blastocyst with intact zona was placed in a 0.5% pronase solution (Sigma P8811) for 10 minutes to remove the zona pellucida. The zona-free blastocysts were then washed in calcium and magnesium-free phosphate-buffered saline (PBS; Gibco, Waltham, MA, USA). Washed blastocysts were exposed to rabbit anti-mouse antibody (Sigma M5774; concentration 1:50) for 30 minutes at 37°C, then washed in calcium and magnesium-free PBS, and transferred into a solution containing: (1) guinea pig complement serum (Sigma S1639; concentration 1:4), (2) 10 μg/mL Hoechst 33342 (Sigma H1399), and (3) 20 μg/mL propidium iodide (Sigma P4170) for 30 minutes at 37°C. The blastocysts were washed, placed on a glass slide, and allowed to air dry. The slides were covered with coverslips and mounted with glycerol. The cell numbers were counted using a fluorescence microscope (Nikon E600) with an excitation filter of 330-385 nm and a barrier filter of 400 nm, and then analyzed using LUCIA Cytogenetics FISH software (Laboratory Imaging, Prague, Czech Republic). The inner cell mass (ICM) nuclei were stained blue with Hoechst, while trophectoderm (TE) nuclei were stained red with propidium iodide.

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.

RNA FISH on mouse embryos was performed as described previously with minor modifications (Borensztein et al., 2017 (link), Ranisavljevic et al., 2017 (link)). Embryos were recovered at E3.5-E4.5 by flushing the uterus with M2 medium (Sigma) and/or by dissection from the uterus. Zona pellucida was removed using acidic Tyrode’s solution (Sigma), and embryos were washed twice with M2 medium (Sigma). ICM was then isolated by immunosurgery, by culturing blastocysts without zona pellucida in anti-mouse red blood cell serum from rabbit (Rockland) for 30 min then in guinea pig complement serum (Sigma) for 15–30 min. For consistency, ICMs from both wild-type and homozygous knockout embryos (from separate crosses) were placed in different regions of the same coverslip before the FISH procedure (Ranisavljevic et al., 2017 (link)).

Differential staining was performed on all hatching and hatched blastocysts, using the protocol described by Pampfer et al. [23 (link)]. In brief, the blastocysts were washed three times in calcium- and magnesium-free buffer, before exposure to rabbit anti-mouse antibody (Sigma M5774; concentration 1:50) for 30 minutes at 37℃. After washing, they were transferred into guinea pig complement serum (Sigma S1639) with propidium iodide (Sigma P4170) and bisbenzimide (Sigma B2261) at 37℃ for 10–15 minutes. The blastocysts were washed and transferred onto glass slides to allow air drying. The slides were mounted in glycerol, and the number of cells in the inner cell mass (ICM) and the trophectoderm (TE) were counted using a Nikon E600 epifluorescence microscope, equipped with the LUCIA FISH program (Laboratory Imaging, Prague, Czech Republic). The ICM nuclei were observed to stain blue, while the TE nuclei showed an intense pink color. Mitotic nuclei were counted as one piece, but dead or pyknotic nuclei were not counted.