Bodipy atp

After IVM, oocytes with the first polar body were selected for staining. Oocytes were fixed in 4% paraformaldehyde at 4 °C for 7 h, then transferred to DPBS containing 500 nM BODIPY-ATP (BODIPY FL ATP; A12410; Molecular Probes, Eugene, OR, USA), 10 μg/mL BODIPY-LD (BODIPY 493/503; D3922; Molecular Probes) or 6 μM BODIPY-FA (BODIPY 558/568 C12; D3835; Molecular Probes) for ATP, LDs, and FAs for 1 h. Next, oocytes were washed thrice with DPBS and transferred to a confocal dish. Lastly, oocytes were photographed using a laser confocal microscope (Leica TCS SP8; Wetzlar, Germany) and ImageJ software (version 8.0.2, NIH, Bethesda, MD, USA) to examine fluorescence intensity. Each group selected at least 40 individual oocytes for result analysis.

Fluorescence staining was performed as described by Jin et al. [18 (link)]. Porcine oocytes with 1st polar body were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C, and then placed into DPBS supplemented with 10 µg/mL BODIPY-LD (BODIPY 493/503; D3922; Molecular Probes, Eugene, OR, USA) for LDs, 6 µM BODIPY-FA (BODIPY 558/568 C12; D3835; Molecular Probes, Eugene, OR, USA) for FAs, or 500 nM BODIPY-ATP (BODIPY FL ATP; A12410; Molecular Probes, Eugene, OR, USA) for 1 h. Following, samples were washed 3 times and mounted on coverslips. Using an epifluorescence microscope (TE2000-S; Nikon, Tokyo, Japan) the stained samples were imaged, and the mean fluorescence intensity of individual oocyte was measured using ImageJ software (version 1.46r; National Institutes of Health, Bethesda, MD, USA). Fluorescence signals were quantified from at least 30 individual oocytes per each group for one biological repetition.