Granulosa cells were collected and washed with PBS three times at room temperature. They were then placed in PBS containing 0.1% Triton X‐100 at room temperature for 30 minutes. After blocking in PBS‐5% bovine serum albumin (BSA) at 37°C for 1 hour, the granulosa cells were incubated with the special antibodies described above at 4°C overnight. Cells were further incubated with goat anti‐mouse and goat anti‐rabbit at 37°C in the dark followed by staining of nuclei with DAPI for 5 minutes. Finally, all granulosa cells were placed on slides with mounting medium (Beyotime) and gently covered with ethanol‐primed coverslips and kept in the dark until confocal scanning.

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