Ecl plus detection kit

MAPK Family Antibody Sampler Kit (#9926) and Phospho-MAPK Family Antibody Sampler Kit (#9910) were purchased from Cell Signaling Technology (Beverly, MA). Anti-CEP-1 (cC-18) (sc-135460) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Paraquat (PQ), Cyclosporin A (CsA), Bongkrekic acid (BA) and 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) were from Sigma. N-acety-L-cysteine (NAC) was purchased from TCI (Shanghai, China). Total Antioxidant Capacity Assay Kit with the ABTS Method (S0119), Fluo-3 AM (S1056), Hoechst 33342 (C1022), Mitochondrial Membrane Potential Assay Kit with JC-1 (C2006), Enhanced BCA Protein Assay Kit (P0010S) and Superoxide dismutase (SOD, S0088) were obtained from Beyotime (Shanghai, China). Acridine orange (AO) was from Dingguo Changsheng Biotechnology (Beijing, China). H₂DCF-DA (D399), MitoSox (M36008), Mitotracker red (M7512), ATP determination kit (A22066) and SYTO 12 (S7574) were purchased from Molecular Probes (Eugene, Oregon, USA). PVDF membranes and ECL plus detection kit were obtained from Millipore (Bedford, MA, USA).

After assessment of neurological defect score, rats (n = 5 per group) were killed by overdose of 10% chloral hydrate (4 ml/kg BW, i.p.) and the ischemic hemisphere was isolated for further Western blot analysis. Each ischemic hemisphere was centrifuged at 12,000 rpm for 30 minutes at 4°C. The supernatant was collected, and protein concentration was measured using a BCA protein assay kit (Beyotime, Shanghai, China). Protein extract and sample buffer were mixed and boiled 5 minutes at 100°C before loading onto 15% polyacrylamide gels. We performed Western blot analysis using standard techniques with an ECL Plus detection kit (Millipore, Billerica, MA). The antibodies used in Western blot analysis were rabbit anti-Bcl-2 (Boster, Wuhan, China, 1 : 200) and mouse β-actin (1 : 1000; ZSGB-Bio). Every sample was repeated 3 times for Western blot analysis. Bands were normalized to β-actin levels, and the density of the band was measured using ImageJ analysis software (NIH, Bethesda, MD). Data were averaged, expressed as the mean 6 SEM, and compared between 2 groups.

The purified histones from serum and tissue that were resolved on 18% sodium duodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was either stained by silver staining method [19 (link)] or transferred to PVDF membrane, probed with site-specific modified histone antibodies, against H4K16Ac (Millipore#07-329), H4K20Me3 (Abcam#9053), γH2AX (Millipore#05-636), H3S10P (Millipore#06-570), H3K27Me3 (Millipore#07-449) and H3K9Me3 (Abcam#8898), and signals were detected by ECL plus detection kit (Millipore #WBKLS0500). Gel loading equivalence was done by silver staining method.