Mercury lamp

Cells on glass cover slips were washed with PBS and fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 20 min. Then cells were blocked with bovine serum albumin (5% in PBS) for 1 h at room temperature, followed by incubating with antibody against CHOP overnight at 4°C. Slides were washed three times with PBS, and incubated with Alexa-488 conjugated goat anti mouse (Invitrogen, Carlsbad, CA, United States) for 1 h at room temperature. Nuclei were counterstained with 2 μg/ml 4′,6-diamidino-2-phenylindole for 1 min. The fluorescent signals were observed under a mercury lamp (Olympus, Tokyo, Japan).

The number of cells in the inner cell mass (ICM) and the trophectoderm (TE) of expanded and hatched blastocysts was determined using differential staining on day 7.5 post-IVF, based on previously published methods46 (link). Briefly, embryos were placed in 0.5% (w/v; 10 μL) pronase in GMOPS (Vitrolife) to remove the zona pellucida, and then washed in GMOPS-PLUS (Vitrolife) for 5 min. Embryos were subsequently incubated in 10 μL of 10 mM trinitrobenzenesulfonic acid (TNBS) in 4 mg/mL polyvinyl pyrollidone (PVP) in simple-G1 medium (Vitrolife; G1-PVP) for 20 min, washed twice in GMOPS-PLUS then transferred to 0.1 mg/mL anti-2,4 dinitrophenol-G1-PVP (10 μL) for 10 min. Following a further wash in GMOPS-PLUS, embryos were transferred to 10 μL drops of 10% v/v guinea pig serum in 25 mg/mL propidium iodide in GMOPS for 1 min, and then placed into 10 μL drops of 0.1 mg/mL bisbenzimide for 20 min. Embryos were individually mounted in 100% glycerol on a glass slide under a coverslip. Embryos were viewed under fluorescent light using a Nikon Eclipse TS100 microscope equipped with a mercury lamp (Olympus) and images of embryos were taken using the Nikon Digital Sight DS-L2 (Nikon). The number of cells in the inner cell mass (ICM) and the trophectoderm (TE) were counted using the cell-counter tool in ImageJ software (National Institute of Health).

Approximately 10⁴ microglial cells were plated per well in a 96-well plate and incubated with Meth for 24 h, then 10 µL Cell Counting Kit-8 Reagent (DojinDo, Japan) was added to each well. After incubation at 37°C for 4 h, absorbance was measured at a wavelength of 450 nm using a microplate reader (TECAN M200,USA). Microglial cells apoptosis was detected by the in situ cell death detection kit, AP (Roche Applied Science, USA) according to the manufacturer's instructions. In brief, 0.2×10⁶ cells were planted on coverslips in 12-well plates, then the cells were incubated with Meth for 24 h, after that, the cells were washed, and fixed with 4% paraformaldehyde, followed by permeabilizing with 0.2% Triton X-100 in ice for 20 min. Then the cells were processed with TUNEL staining (Green). ProLong Gold antifade reagent (Molecular Probes, USA) with 40, 60-diamidino-2-phenylindol (DAPI) was added to stain cell nuclei. Cells were visualized by fluorescence microscope (Olympus IX70, Japan), adapted with a Mercury lamp (Olympus, Japan).The apoptosis cells were counted and normalized to the total number of nuclei stained with DAPI.