Anti cleaved parp

The following reagents and antibodies were used: Bongkrekic acid (Santa Cruz Biotechnology, La Jolla, CA), JC-1 (eBioscience, San Diego, CA), Cyclosporine A, 2′,7′-dichlorofluorescein acetate (DCFH-DA), N-acetyl cysteine (Sigma, St.Louis, MO), Dioleylphosphatidylserine (Avanti Lipids, Alabaster, AL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) ((Roche Diagnostics, Indianapolis, IN), and disuccinyl suberate (Thermo Scientific Fischer, Rockford, IL). Anti-Bcl-2, anti-β-Actin (Abcam, Cambridge, MA), anti-Cyto c (eBioscience, San Diego, CA), anti-AIF, anti-caspase-3, anti-cleaved caspase-3 (Cell Signaling Technology, Boston, MA, anti-Survivin, Smac/Diablo, α-Tubulin (Novus biological, Littleton, CO), anti-COX-4, anti-Bax (N-20; Santa Cruz Biotechnology, La Jolla, CA), anti-Bax (polymer-recognizing A67 clone; Sigma, St.Louis, MO) and anti- cleaved PARP (Millipore, Bedford, MA).
Animal maintenance and experimental procedures were carried out in accordance with the US National Institute of health Guidelines for Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and Cincinnati Children's Hospital Medical Center.

Excised tumors, kidney and livers were sampled just after sacrifice and representative areas were a) formalin-fixed (24 hours) (Millipore) and paraffin-embedded and (b) snap-frozen in OCT and stored at 80ºC as previously described [32 (link)]. Tissue sections 2μM thick were stained with hematoxilin & eosin and prepared for IHC. Two experienced pathologists (MCGM and EDA) observed the samples under a Leica microscope (Leica Microsystems). IHC was performed as previously described [32 (link)] using the following primary antibodies: anti-pH2AX (Millipore); anti-cleaved PARP (cell signaling); anti-Ki67 (Millipore) and anti-BRCA2 (Millipore). TUNEL assays (Roche) were performed to detect DNA fragmentation and late apoptosis. To quantify the extent of necrosis and the IHC findings, the Dotslide analysis program (Olympus) and the Ariol Image analysis system (Olympus) were used respectively.