Goat anti rabbit igg secondary antibody

EAT sections embedded in paraffin were dewaxed, hydrated with xylene, and immersed in EDTA antigen retrieval (pH 8). Specimens were then blocked with tris-buffered saline (TBS) and 5% bovine serum albumin (BSA) at room temperature for 1 h, followed by incubation with primary antibody specific to caspase-3 over night at 4 °C. Slides were afterwards washed with TBS and incubated with goat anti-rabbit IgG secondary antibody (Servicebio, China) for 30 min at room temperature. Sections were then washed with TBS and visualized using diaminobenzidine (DAB) (Liquid DAB + Substrate Chromogen System; Dako, Denmark). Mayer's hematoxylin (Sigma-Aldrich, Inc. USA) was used finally for counterstaining. Percentage (%) of caspase-3 immunopositive areas (distinct brown color) to total area was determined through selecting 5 non-overlapping fields (magnification ×100) in each section (mean ± S.E.M of 5 fields). Stained tumor sections images were analyzed using full HD microscopic imaging system linked to Leica application suite (Leica Microsystems GmbH, Germany).

NaHS was obtained from Sigma Chemical (St. Louis, MO, USA); IPTG (catalogue number: G5042-5G), goat anti-rabbit IgG secondary antibody (catalogue number: G1213-100U), anti-GAPDH antibody (catalogue number: GB12002), marker (catalogue number: G2058-250UL), Coomassie blue R250 (catalogue number: GM1002), LDH assay kit (catalogue number: G1610-100T) were purchased from Servicebio (Wuhan, China); NSE assay kit (catalogue number: MM-0069R2) was obtained from Jiangsu Meimian Industrial, Co., Ltd. (Jiangsu, China); anti-ROCK₂ antibody (catalogue number: ab125025), anti- ROCK₂ (phospho Y722) antibody (catalogue number: ab182648), ATP (catalogue number: ab181719), recombinant Src (catalogue number: ab79635) were purchased from Abcam (San Francisco, CA, USA); 293T cell (catalogue number: c6008) was purchased from Beyotime Biotehchnology, Co. (Shanghai, China).

Cells were washed with ice-cold PBS and lysed with RIPA buffer. Samples containing equal quantities of total proteins were resolved on 15% SDS polyacrylamide denaturing gel and transferred to nitrocellulose membranes. After blocking with 5% skim milk in TBST for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies: AKT (1 : 1000, Abcam, UK), JNK (1 : 1000, Abmart, Shanghai, China), and p38 (1 : 1000, Abmart, Shanghai, China). Goat anti-rabbit IgG secondary antibody (1 : 5000) (Servicebio, Wuhan, China) was used at room temperature for 1-h incubation. The blot was visualized by using ECL Chemiluminescence Kit (Epizyme, Shanghai, China). Each band was quantified via ImageJ software.