Anti cdx2 antibody

Embryos were fixed overnight at 4°C in immunostaining fixing solution (Beyotime, P0098). Unless otherwise stated, all staining steps were performed at room tem-perature. Embryos were permeabilized with immunostaining solution containing Triton X-100 (Beyotime, P0096) for 0.5 h. After washing 5 times, they were blocked at 4°C for 12 h in the immunostaining blocking solution (Beyotime, P0260) and then incubated with primary antibody at 4°C for 12 h. Anti-CDX2 antibody (BioGenex) was diluted 1:50 using QuickBlock dilution buffer for immunostaining (Beyotime, P0262). After washing 5 times, embryos were incubated with secondary antibody Alexa Fluor 555-labeled goat antimouse IgG (Beyotime, A0459) for 2 h, washed, and then sealed with 4,6-diamidino-2-phenylindole (DAPI; Beyotime, C1005), 10 min, sealing tablets (Su et al., 2012) (link). Finally, the embryos were photographed with a LSM 700 fluorescence microscope (Carl Zeiss). All experiments were replicated at least 3 times with a group of 10 to 15 embryos in each replicate. For fluorescence quantification, the intensity levels of the NT group and ZFP57 overexpression/knockdown embryos were expressed relative to the average intensity level of IVF embryos. Intensities were measured as described previously (Zhang et al., 2022b) .