Dcfh diacetate dcfhda kit

Mouse polyclonal anti-DRP1 antibody was purchased from Abcam (Cambridge, UK). Hoechst 33342 was purchased from Sigma (St. Louis, MO, USA). Goat anti-rabbit IgG/FITC and goat anti-rabbit IgG/TRITC were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Basic maturation culture medium was tissue culture medium (TCM-199) (Sigma). Mito-Tracker Red CMRos (1:500, Cat# M7521, Invitrogen, Eugene, OR, USA) was used to detect the distribution of mitochondria with an ultimate density of 2 μmol/L. The DCFH diacetate (DCFHDA) kit (Beyotime, Beijing, China) was purchased to determine the ROS levels in living oocytes. Annexin-V-FITC (Yeasen Biotech Co., Ltd., Shanghai, China) was used to determine apoptosis levels. Mdivi-1 was purchased from MedChemExpress (Shanghai, China).

To determine the level of ROS in living embryos DCFH diacetate (DCFHDA) kit (Beyotime, China) was used. Embryos were incubated in PZM-3 medium with DCFHDA (1:800) for 30 min at 38.5°C in 5% CO₂ incubator. After wash embryos three times and fluorescent signals examined with microscope (CKX53, Olympus, Japan). Embryos were incubated in PZM-3 medium with Mito-Tracker Red CMXRos (1:200) (Cat #M7512, Invitrogen, Eugene, OR, United States) at 38.5°C for 30 min. Then wash the embryo three times with the PZM-3 culture medium and examined with a confocal laser-scanning microscope (Zeiss LSM 700 META, Germany).

A DCFH diacetate (DCFHDA) kit (Beyotime, China) was used to examine the level of intracellular ROS generated during oocyte maturation. Mito-Tracker Red CMXRos (Invitrogen, Eugene, OR, United States, #M7512) was used for mitochondria detection. After DOs were obtained, they were incubated in TCM-199 culture medium containing DCFHDA (1:800) or Mito-Tracker Red CMXRos (1:200) for 30 min at 38.5°C in a 5% CO₂ incubator. After three washes in TCM-199, oocytes were placed on a glass slide and observed under the confocal laser-scanning microscope as soon as possible.
As for mitochondrial membrane potential (ΔΨm) detection, MitoProbe JC-1 Assay kits (M34152, Thermo Fisher Scientific, Waltham, MA, United States) was used. The steps of JC-1 staining are the same as above and 2 μM JC-1 was added in TCM-199 culture medium. The change in JC-1 from red (∼590 nm) to green (∼529 nm) fluorescence was used to detect a decrease in mitochondrial membrane potential. The ratio of red to green fluorescence intensity was analyzed using ImageJ software.