Cyanine3 goat anti rabbit

All blastocysts were treated by 5mg/mL pronase (Sigma) at 37 C (5% CO2, air) for 5-10 min to remove zona pellucida, and then washed in M16 (Sigma) and fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min. After that, all fixed blastocysts were washed by PBS and permeabilized in 1% Triton X-100 in PBS at room temperature for 30 min, and blocked in 0.1% Tween-20, 0.01% Triton X-100 and 1% BSA in PBS at room temperature for 1 hour. Blocked blastocysts were incubated with primary antibodies (CDX2 and GAB1/JADE1/SMOC1) at 4 C overnight. Secondary antibodies were incubated at room temperature for 1 hour. Used secondary antibodies included Alexa 488 donkey anti-mouse (Invitrogen) and Cyanine3 goat anti-rabbit (Invitrogen). DNA contents were stained by DAPI (4 0 ,6-diamidino-2-phenylindole, 5 mg/ml, Beyotime) incubation at room temperature for 5 min. To compare the extraembryonic protein levels of H3K27me3-dependent imprinting genes, the relative fluorescence strength of GAB1, JADE1, and SMOC1 in dozens to hundreds of trophectoderm (TE) cells from the IVF, WT-NT, and D4-NT blastocysts were analyzed. TE cells with positive CDX2 signals were chosen for analyzing. Specific values of GAB1/JADE1/SMOC1 protein/DAPI signal ratios in each chosen TE cell were recorded (Leica SP8) and analyzed (IMARIS 3D/4D Visualization & Analysis Software).