Bcl 2 antibody

Samples for western blotting were collected from brain infarction boundary regions. After extraction and quantification, equal amounts of protein were separated by sodium dodecyl sulfate-PAGE and then transferred to an immobilon-polyvinylidene fluoride membrane. The membranes were blocked using 5% milk for 2 h at room temperature and probed with the primary antibody overnight at 4°C. These primary antibodies included rabbit anti-phospho-ERK1/2 (1 : 5,000), rabbit anti-total-ERK1/2 (1 : 5,000), rabbit anti-phospho-cAMP response element-binding protein (CREB) (1 : 1,000), rabbit anti-total-CREB (1 : 1,000), rabbit anti-Bcl-2 (1 : 200), rabbit anti-Bax (1 : 400), rabbit anti-cleaved caspase-3 (1 : 1000), and rabbit anti-β-actin (1 : 5,000). The secondary antibody was anti-rabbit HRP-conjugated antibody (1 : 4,000). All primary antibodies were purchased from Cell Signaling Technology (Danvers, USA), with the exception of the Bcl-2 antibody purchased from Santa Cruz (Dallas, USA). Immunoblots were developed with the Immobilon ECL method (Millipore, Billerica, Massachusetts, USA). Gray bands were converted to density values using Image J software for quantification analysis.

In this study was used: anti-rabbit and anti-goat secondary antibodies as well as polyclonal RIP1, RIP3, Bax and Bcl-2 antibody from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal LC3 antibody from Abcam; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and RNAse A and 2′,7′-Dichlorofluorescin diacetate (DCF-DA) from Sigma-Aldrich; Annexin V-FITC apoptosis detection kit and propidium iodide staining solution from BD Pharmingen; BrdU incorporation ELISA assay from Roche (Grenzach-Wyhlen, Germany).

Hepatic and renal specimens were cut into slices, dehydrated, and embedded in paraffin for histological analysis. Slices were then cut into 3 μm thick to be stained with hematoxylin and eosin (H&E), then visualized under an optical microscope.
Slices were embedded in paraffin, deparaffinized, and remoistened for immunohistochemical analysis. They were then cleaned in PBS and immersed for 15 min in 2 percent H2O2 to impede peroxidase activity. Bovine serum albumin (5%) was used to block non-specific binding sites. Bcl-2 Antibody [(C-2): sc-7382], Caspase-3 Antibody [(9CSP01): sc-81,663], and polyclonal antibodies (Santa Cruz Biotechnology, USA) were employed to coat the kidney and liver tissue specimen slides before being diluted to 1:500 and added. The slides were then kept at 4 °C for overnight incubation. A biotin-conjugated secondary antibody (catalog # sc-2040) was implemented to the slides after three PBS rinses. These were created with 3,3-diaminobezidine tetrahydrochloride, and hematoxylin was used as a counterstain [44 (link)]. The relative proportions of immune reactive cells for caspase-3 and Bcl2 measured by the ratio of positively stained cells to the total number of examined cells. Three slides from six rats in each group were the subjects of ANOVA tests to determine their significance.

Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 0.2 mM PMSF) and proteins (20–40 μg) were isolated and separated on 8–10 % SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking procedure, membranes were incubated with primary antibodies, followed by HRP-conjugated secondary antibodies, and then the proteins of interest were visualized using the Imager (Bio-Rad) and ECL (Thermo Fisher Scientific, Rochester, NY) system. Antibody of GAPDH was purchased from Abcam (Cambridge, MA) and antibodies of Mcl-1and cleaved caspase-3 were obtained from Cell Signaling (Danvers, MA). Bcl-2 antibody was obtained from Santa Cruz (Santa Cruz, CA).

(+)-JQ1 was purchased from ApexBio (Houston, TX). OTX015 and MS436 were purchased from Cayman Chemical (Ann Arbor, MI). Soluble recombinant human TRAIL was purchased from PeproTech, Inc. (Rocky Hill, NJ). MG132, cycloheximide (CHX) and PFI-1 were purchased from Sigma Chemical Co. (St. Louis, MO). Monoclonal anti-FLIP antibody (NF6) was obtained from Alexis Biochemicals (San Diego, CA). Mouse monoclonal caspase-8, PARP, survivin and c-Myc antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Mouse monoclonal caspase-3 antibody was purchased from Imgenex (San Diego, CA). Rabbit polyclonal DR5 antibody was obtained from ProSci Inc. (Poway, CA). Mouse monoclonal DR4 antibody (B-N28) was purchased from Diaclone (Stamford, CT). Rabbit polyclonal Mcl-1 and Bcl-X_L/S and mouse monoclonal Bcl-2 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Tubulin and GAPDH antibodies were purchased from Sigma Chemical Co. and Trevigen Inc. (Gaithersburg, MD), respectively.