The cells with clear nuclear labeling were defined as TUNEL positive cells. The apoptotic index was calculated as the number of TUNEL positive cells/ total number of myocytes 100. Immunohistochemistry Transmural sellekchem LV samples were embedded in paraffin and cut into 5um sections. Detection of Bcl 2 expression was performed as described previously. Tissue sec tions were exposed overnight to rabbit polyclonal anti bcl 2 antibody at 4 C, washed in PBS and then incubated with bio tinylated goat antirabbit IgG for 60 min at 37 C. After two washing steps, sections were exposed to streptavi din horseradish peroxidase complex for 30 min at 37 C, and then visualized with 3,3 diaminobenzidine, embedded in glycerol gelatin and coverslips applied. Images were captured digitally and analyzed using Image Pro Plus version 6.

0. The result was expressed as the ratio of positive to negative staining area. Western blot analysis Transmural LV samples were obtained after 10 min of reperfusion. Western blotting was performed as described previously. In brief, freshly frozen myo cardial tissue samples were homogenized in RIPA buf fer. Total protein was separated Inhibitors,Modulators,Libraries by 10% SDS PAGE and transferred to nitrocellulose membranes. Mem branes were exposed to p Akt, Akt, p STAT3 or STAT3 antibody, and subsequently incubated with a chemilumi nescence substrate and exposed to radiographic film. The images were captured digitally and the density at specific molecular weights was measured using Gel Pro analyzer version 3. 0. Quantitative reverse Inhibitors,Modulators,Libraries transcriptase polymerase chain reaction Bcl 2 mRNA levels were determined by quantitative Inhibitors,Modulators,Libraries reverse transcriptase polymerase chain reaction.

For extraction of total RNA the RNeasy Mini Kit was used according to the manufacturers instructions. cDNAs were synthe sized using RevertAid First Strand cDNA Synthesis Kit. qPCR was performed with Maxima SYBR Green qPCR Master Mix, ROX Solution Inhibitors,Modulators,Libraries provided. Fluorescent signals were normalized to an internal reference, and the threshold cycle was set within the exponential phase of the PCR. The relative gene expression was calculated by comparing cycle times for each target PCR. The target PCR Ct values were normalized by subtracting the GAPDH Ct value, which provided the Ct value. mRNA levels were quan tified with the 2 relative quantification method.

The following primers were used for detection Bcl 2 Statistical analysis All data are expressed as mean SEM and analyzed using SPSS 15. 0. Independent samples t test and one way ANOVA were used to compare data with post hoc analysis using the Student Newman Keuls correction. Inhibitors,Modulators,Libraries selleckchem Ganetespib A p value 0. 05 was considered significant. Results Hemodynamic data There were no baseline differences. After 30 min ische mia, mean arterial blood pressure decreased significantly between the sham and intervention groups. No statisti cal differences were observed among all groups at both 2 and 24 hours after reperfusion.