4.4. Immunofluorescence Analysis and Antibodies

Confocal microscopy was performed to determine protein expression and localization in the samples [52]. COCs, mature oocytes (MII)/zygotes/two-cell/four-cell/eight-cell/16-cell stage embryos, and day-8 blastocysts were collected from the control activator and inhibitor groups. The collected embryos were fixed with 4% (w/v) paraformaldehyde in 1.0 M PBS. Fixed embryos were washed thrice with polyvinyl alcohol (0.3%) in 1× PBS (PVA PBS) for 15 min. Proteinase K was added to the samples and incubated for 5 min to enhance permeability. The samples were washed three times with PVA in PBS for 15 min and incubated with the blocking solution (BSA 5% in PBS/PVP) at 27 °C for 90 min. Then, the samples were incubated overnight with PDGFRβ (Santa Cruz Biotechnology Inc., Dallas, TX, USA; cat. # sc-373805), NFYA (My Biosource.com, San Diego, CA, USA, cat. MBS822279) primary antibodies at 4 °C. The samples were thoroughly washed the following day with PVA-PBS for 15 min. Thereafter, the samples were incubated with TRITC and FITC secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at 27 °C for 90 min and washed thrice with PVA PBS for 15 min to remove residual stains. To stain nuclei, 4, 6-diamidino-2-phenylindole (DAPI, 10 g/mL) was added to the samples at 27 °C for 5 min. After washing, all samples were fixed on slides. A laser scanning confocal microscope (Fluoview FV 1000; Olympus, Tokyo, Japan) was used for confocal imaging. Signal intensities were measured using ImageJ 154 software (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij, accessed on 1 September 2022).