Cell counting and TUNEL assay

Mice embryos at the 96h-stage of development, which were morphologically compacted morulae or blastocysts, were collected to detect apoptosis between the control and experiment groups. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used by an In Situ Cell Death Detection Kit (Roche, 11684795910, USA) according to the manufacturer’s protocol. Briefly, after fixing in 4% PFA then permeabilizing in 0.5% triton X-100, TUNEL reaction mixture was used to label DNA strand breaks, and DAPI was subsequently used to show the nuclei of embryos. The images of embryos were captured in depth by Olympus FLUOVIEW FV1000 laser confocal microscope (Olympus, Japan), and they were used for the cell counting by two individuals who were unware of the group information of treatments.