Ix83 spinning disc confocal microscope

After culturing and removing cumulus cells, oocytes and blastocysts were fixed for 30 min in 4% paraformaldehyde and treated with fluorescein-conjugated dUTP and the terminal deoxynucleotidyl transferase enzyme (In Situ Cell Death Detection Kit, cat. No. 11684795910, Roche, Germany) for 60 min at 38°C in the dark, in accordance with the assay protocol. A positive control was prepared in accordance with the manufacturer´s instructions. Finally, after incubation, oocytes and/or blastocysts were washed three times in PBS and mounted onto slides in Vectashield medium with DAPI. Oocytes and apoptotic cells with a positive fluorescence signal were considered TUNEL-positive cells. Images of oocytes were acquired using an Olympus IX83 spinning disc confocal microscope (Olympus, Germany). Based on DAPI observation, the fragmentation of nuclei and total nuclei in blastocysts wereobserved under a fluorescence microscope (Nikon Eclipse Ci microscope, Nikon Instruments Inc., Seoul, Korea).

The mitochondrial distribution and shape of clusters were evaluated using MitoTracker Red (Thermo Fisher Scientific), which selectively stains live mitochondria. Cumulus cell-free oocytes after 48 h of IVM were incubated with 200 nM MitoTracker diluted in M199 maturation medium at 38°C for 30 min in 5% CO₂ in air, washed 3 times and mounted in Vectashield containing DAPI. Images were acquired using an Olympus IX83 spinning disc confocal microscope (Olympus, Germany).

The trimmed blocks were mounted in a drop of PBS in the center of a glass-bottom imaging plate (Cellvis; D35-10-1.5N) and fixed to position with plasticine. Blocks were imaged with an Olympus spinning disc confocal IX83 microscope (details under “Confocal image acquisition”). Iterative imaging and polishing were performed in cases where the regions of interest were more than ∼8 µm deep from the polished surface.