Rnasin plus rnase inhibitor

Frozen midbrain samples were homogenized in 1 ml ice-cold homogenization buffer [320 mM sucrose, 5 mM CaCl₂, 3 mM Mg(Ac)₂, 10 mM Tris pH7.6, 0.1 mM EDTA, 0.1% NP40, 0.1 mM PMSF, 1 mM β-mercaptoethanol, 1% BSA, 1:250 RNasin Plus RNase Inhibitor (Clontech)] using a 1 ml Dounce homogenizer (Wheaton); 30 times with pestle A, followed by 30 times with pestle B. After 10 min on ice, the homogenate was filtered with 40 μm cell strainer (Fisher) and added 1 ml dilution buffer [50% OptiPrep density gradient medium (Sigma), 5 mM CaCl₂, 3 mM Mg(Ac)₂, 10 mM Tris pH 7.6, 0.1 mM PMSF, 1 mM β-mercaptoethanol] and mixed thoroughly with pipette. Loaded 0.5 ml lysate on the top of 0.5 ml 29% iso-osmolar OptiPrep solution (in PBS) in a 1.5 ml centrifuge tube and centrifuged at 6000×g for 10 min at 4 °C. After removing the supernatant, the nuclei were resuspended in wash buffer [2.5 mM MgCl₂, 1% BSA in PBS, 1:500 RNasin Plus RNase Inhibitor (Clontech)] for immunostaining or FANS.