The pups were sacrificed by decapitation at 24 h after HI. Tissue from the parietal cortex (including the hippocampus) in both hemispheres was rapidly dissected out and homogenized immediately on ice using a 2-ml Dounce tissue grinder set (Sigma, D8938), and an isolation buffer was added (15 mM Tris–HCl, pH 7.6, 320 mM sucrose, 1 mM dithiothreitol, 1 mM MgCl2, 3 mM EDTA-K, and 0.5% protease inhibitor cocktail (Sigma, P8340)). The procedures for cellular fraction isolation, including nuclear, cytosolic, and mitochondrial fractions, were as described in our previous study [30 (link)]. All fractions were kept at – 80 °C.
Li T., Sun Y., Zhang S., Xu Y., Li K., Xie C., Wang Y., Wang Y., Cao J., Wang X., Penninger J.M., Kroemer G., Blomgren K, & Zhu C. (2022). AIF Overexpression Aggravates Oxidative Stress in Neonatal Male Mice After Hypoxia–Ischemia Injury. Molecular Neurobiology, 59(11), 6613-6631.
Other organizations :
Zhengzhou Children's Hospital, Zhengzhou University, Third Affiliated Hospital of Zhengzhou University, University of Gothenburg, Austrian Academy of Sciences, Institute of Molecular Biotechnology, Centre de Recherche des Cordeliers, Université Paris Cité, Inserm, Institut Universitaire de France, Sorbonne Université, Karolinska University Hospital
Isolation buffer composition (15 mM Tris–HCl, pH 7.6, 320 mM sucrose, 1 mM dithiothreitol, 1 mM MgCl2, 3 mM EDTA-K, and 0.5% protease inhibitor cocktail)
Cellular fractionation procedure (as described in previous study)
controls
No positive or negative controls were explicitly mentioned in the provided information.
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