Incubation chamber stage

The membrane-sensitive dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide) was used to detect mitochondrial membrane potential (ΔΨ m) according to the vendor’s instruction with some modifications (10009172; Cayman Chemical Company). Briefly, the JC-1 stock solution was diluted (x40) in the KSOMaa medium (KSOM + JC1). Embryos injected with siRNAs against Gabpb1 or control siRNAs were incubated in KSOM + JC1 for 30 min at 37°C in air containing 5% CO₂. After the incubation, embryos were washed three times in phenol red-free KSOMaa medium containing 0.00025% PVA (KSOM + PVA). Then, embryos were transferred to drops of KSOM + PVA on a glass-bottom dish (Matsunami) and placed in an incubation chamber stage (Tokai Hit) at 37°C under 5% CO₂ in air for live-cell imaging. The fluorescence signals were observed using an LSM800 confocal microscope, equipped with a laser module (405/488/561/640 nm) and GaAsP detector, using the same contrast, brightness, and exposure settings (3 μm interval). For detecting J-aggregates and monomers, the rhodamine and FITC filter settings were used, respectively. Membrane potential is expressed as a ratio of the average rhodamine fluorescence (J-aggregates) relative to the average FITC fluorescence (monomers) in embryo areas.