Horseradish peroxidase hrp conjugated secondary antibody

Differences in KSC and RTEC protein expression were determined by western blotting. For this, cell protein was extracted using RIPA buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail [Set I, Calbiochem, USA]). Extracts were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (PVDF, Bio-Rad). Membranes were blocked with TBST containing 5% skim milk and incubated with primary antibodies overnight at 4°C. After washing with TBST, membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Beijing Zhongshan Biotechnology, Beijing, China) for 1 h at room temperature. After three additional washes with TBST, reactive bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA). The antibodies used for western blots were obtained as follows: rabbit polyclonal OCT4 antibody (11263-1-AP, Proteintech), rabbit polyclonal SOX2 antibody (11064-1-AP, Proteintech), and mouse monoclonal β-actin antibody (A1978, Sigma).

150 bovine oocytes at the MI stage were harvested and washed three time in PBS buffer, proteins were extracted and seperated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using 7.5% polyacrylamide gel and tranferred onto membranes (Millipore). Next, the membranes were blocked in 5% (w/v) skim milk overnight at 4°C. Membranes were incubated with p-MAPK monoclonal antibody (1:2000) and caspase-3 polyclonal antibody (1:1000) at 4°C overnight, washed three times (5 min each time) in TBS, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Zhong Shan Biotechnology, Beijing, China) for 1 h at room temperature. The signals were detected using an ECL kit.

The nitrotyrosine content was also measured by immunohistochemistry staining. The detailed protocol was described in our previous publications [21 (link), 22 (link)]. The LV tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Then, they were cut into 3 mm thickness and stained with primary antibody (anti-nitrotyrosine antibody, Cell Signaling Technology, MA, USA, 1 : 200 dilution). Then, the sections were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (Zhongshan Biotechnology, Beijing, China) and detected with 3,3′-diaminobenzidine (DAB) staining (Zhongshan Biotechnology, Beijing, China). Five fields of each section were randomly chosen and photographed at ×200 magnification (Olympus BX-63, Tokyo, Japan). The graphs were analyzed and calculated using Image-Pro Plus software (Media Cybernetics, MA, USA).

Frozen liver tissues and cultured cells were homogenized in RIPA lysis buffer (Biyotime, Haimen, China) in the presence of 1% (v/w) protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL). The mixture was placed on a shaker at 4°C for 1 h and insoluble matter was removed by centrifugation at 40000× g at 4°C for 1 h. Protein concentration was determined by the Bradford method using bovine serum albumin (BSA) as the standard. Proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membrane was incubated overnight with primary antibodies to LC3-II, p38 mitogen activated protein kinase (MAPK), p-ERK(Novus Biologicals, Littleton, CO), or HO-1(Abcam, Cambridge, UK). β-actin expression was used as a loading control(Abcam). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1∶4000; Beijing ZhongShan Biotechnology, Beijing, China) for 1 h and visualized using an enhance chemiluminesence detection kit following the manufacturer's instructions (Pierce-Thermo Scientific, Rockford, IL). All proteins were detected in more than three independent experiments, and for each protein, the gray value was measured by Image-Pro Plus 6.0 in order to conduct statistical analyses.