2.4. Immunofluorescence Staining of Oocytes and Blastocysts

For oocyte immunofluorescence staining, oocytes were fixed in 4% paraformaldehyde containing 0.1% polyvinyl alcohol (PVA) in phosphate-buffered saline (PBS) for 1 h at 4 °C. Fixed oocytes were washed three times with PBS for 10 min each, transferred onto a glass-slide, and then treated with 0.2% Triton X-100 in PBS for 10 min for permeabilization. Nonspecific staining was blocked using Protein Block Serum-Free (Dako, Carpinteria, CA, USA) for 1 h, followed by incubation with the primary antibodies α-tubulin (1:100, ab15246, Abcam, Cambridge, UK), and pericentrin (PCNT; 1:100, 611814, BD Biosciences, San Jose, CA, USA) overnight at 4 °C. Oocytes were labeled with Alexa Fluor^® 488 and Alexa Fluor^® 594 conjugated secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The nuclei were stained with DAPI (1:1000, 62248, Thermo Fisher Scientific, Fair Lawn, NJ, USA). Oocytes were mounted using Gel Mount Aqueous Mounting Medium (M01, Sigma-Aldrich) and observed using a confocal microscope (Zeiss LSM880, Carl Zeiss, Oberkochen, Germany).

For blastocyst staining, blastocysts were fixed 6 days after IVF, and permeabilization and blocking were performed sequentially. Blastocysts were labeled with a primary OCT4 antibody (1:100, 611203, BD Biosciences), and Alexa Fluor^® 594 conjugated secondary antibody (1:1000, Invitrogen), followed by counterstaining with DAPI to label the nuclei. The number of trophectoderm (TE) cells was calculated as the total cell number minus the inner cell mass (ICM) cell number.