This kind of damage is characterized by single and double DNA strand breaks

n digital camera, and the images were visualized in a computer monitor. For the quantification of LC3-II immunostaining, 10 microscopic fields in each ” section across ischemic hippocampus “9184477 regions in the ipsilateral hemisphere were analyzed. Three sections were used for each animal. The number of cells with LC3-II immunoreactivity in each field was counted by an examiner who was blind to the experimental conditions. removed the brain tissues from ischemic hippocampus area and the corresponding area of sham-operated rats. We immediately placed all of the tissue into dry ice-cold collecting tubes and stored them at -80uC until further analysis. The PC12 cells were cultured in 60-mm dishes and harvested and rinsed twice with ice-cold PBS after OGD. We later homogenized these tissue samples and cells in cold Radio Immunoprecipitation Assay lysis buffer with a 1% proteaseinhibitor cocktail, followed by centrifugation at 14,0006g for 10 min at 4uC. We determined the protein concentration using a BCA protein assay kit. After heating the aliquots of protein in SDSPAGE protein loading buffer at 95uC for 10 min, we separated them on SDS-PAGE gels and transferred the proteins to PVDF membranes for immunoblotting. We incubated the membranes in blocking buffer for 1 h at room temperature, followed by an overnight incubation 4uC with primary antibodies against class III PI3K, Beclin-1, LC3, and Bcl-2. We then washed off the primary antibody three times in TBS, incubated the membranes with horseradish peroxidase-conjugated anti-rabbit IgG antibody for 2 h at room temperature, and washed them three times in TBS. We detected the immunoreactive blots with enhanced chemiluminescence and visualized them on X-ray film. GAPDH was used as the loading control. The signal intensity of primary antibody binding was quantitatively analyzed with Sigma Scan Pro 5 and was normalized to a GAPDH loading control. The statistical analyses were purchase R-roscovitine performed by a one-way analysis of variance followed by the Tukey test. The differences were considered significant when p, 0.05. Statistical Analysis We analyzed the data using SAS software and reported the results as the mean6SD. We analyzed the variance in neuronal damage and the number of LC3-II-positive cells in rat hippocampal pyramidal neurons at a given testing time using a one-way ANOVA. For the between-group variance in the ultrastructural changes and the immunoblot analyses of the PC12 cells or rat hippocampal pyramidal neurons at a given testing time, we performed an ANOVA followed by the Tukey test. We considered a result statistically significant when P,0.05. Author Contributions Conceived and designed the experiments: DC LW WJ. Performed the experiments: DC AQ QZ XZ. Analyzed the data: DC LW. Contributed reagents/materials/analysis tools: DC AQ XZ. Wrote the paper: DC LW WJ. Protein Preparation and Immunoblotting We deeply anesthetized the rats with an overdose of pentobarbital and subsequently cut and quickly References 1. Peters CE, Korcok J, Gelb AW, Wilson JX Anesthetic concentrations of propofol protect against oxidative stress in primary astrocyte cultures: Comparison with hypothermia. ANESTHESIOLOGY 94: 313321. Velly LJ, Guillet BA, Masmejean FM, Nieoullon AL, Bruder NJ, et al. Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: Role of glutamate and its transporters. ANESTHESIOLOGY 99: 368375. 3. Qi S, Zhan RZ, Wu C, Fujihara H, Taga K, et al. The effects of thiopental and propofo