Anti cd86 13395 1 ap

After the experiment, the brain tissues and pancreas tissues of rats were collected, and the total proteins were extracted by radio immunoprecipitation analysis (RIPA) and lysis buffer solution. The protein concentration was determined by bicinchoninic acid (BCA) method. A total of 200 μg protein samples were separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were transferred to a polyvinylidene fluoride membrane that had been activated by methanol and blocked by 5% skim milk and dried at room temperature for at least 1 h. The primary antibodies were then incubated overnight at 4°C. The primary antibodies for incubation included anti-CD11b (66519-1-Ig, 1:2000, Proteintech, USA), anti-CD86 (13395-1-AP, 1:1000, Proteintech, USA), anti-cleaved-caspase 3 (19677-1-AP, 1:1000, Proteintech, USA), anti-p21 (10355-1-AP, 1:1000, Proteintech, USA), anti-P16 (10883-1-AP, 1:1000, Bioss, China), and anti-b-actin (60008-1-Ig, 1:5000, Proteintech, USA). They were then incubated with anti-mouse IgG (SA00001-1, 1:5000, Proteintech, USA) and antirabbit IgG (SA00001-2, 1:6000, Proteintech, USA) at 37 °C for 90 min. Chemiluminescence (Millipore, USA) was visualized and analysed using imaging software (GE Healthcare, Life Sciences, USA).