Anti mouse jnk

Brain tissues were immobilized in 4% paraformaldehyde solution for 24 hours and then dehydrated in 25% sucrose solution. The above specimens were embedded, sliced at 6 μm and stored at −20°C. The frozen sections were washed with PBS after rewarming. Serum was blocked and incubated with antibodies, including anti-rabbit Bcl-2 (Abcam, 1:250), anti-rabbit Bax (Abcam, 1:250), anti-rabbit cleaved Caspase-9 (Cell Signaling, 1:800), anti-rabbit cleaved Caspase-9 (Cell Signaling, 1:500), anti-rabbit cleaved Caspase-3 (Abcam, 1:100), anti-rabbit LC3 (Abcam, 1:2000), anti-rabbit p62 (Proteintech, 1:50), anti-rabbit Beclin1 (Abcam, 1:100), anti-rabbit ASK1 (Abcam, 1:50), rabbit anti-phospho-ASK1 (Thermo Fisher, 1:50), anti-mouse JNK (Proteintech, 1:500), rabbit anti-phospho-JNK (Abcam, 1:100), and anti-mouse β-actin (Proteintech, 1:2000), for 16~18 hours at 4°C. Each section was washed with PBS and then incubated with the proper secondary antibody (Abcam, 1:5000) for 30 minutes. It was visualized by staining with 3,3′-diaminobenzidine (DAB) (Bioss Biotechnology Company, Woburn, MA, USA) kit, and then observed under an optical microscope and photographed. Image visualization used Image-Pro Plus 6.0 based on the integral optical density, which reflects the change of optical density and area of the positive substance, i.e., the total antigen level.

RIPA lysis buffer was used to extract proteins from brain tissues, and then the bicinchoninic acid protein assay kit (Beyotime, China) was used to determine the protein concentration. Equal amounts of protein were subjected to 10% SDS-PAGE prior to transfer onto PVDF membranes (Millipore). After being blocked with 5% nonfat milk, the membranes were then incubated with proper antibodies, including anti-rabbit Bcl-2 (Abcam, 1:1000), anti-rabbit Bax (Abcam, 1:1000), anti-rabbit cleaved Caspase-9 (Cell Signaling, 1:1000), anti-rabbit cleaved Caspase-8 (Cell Signaling, 1:1000), anti-rabbit cleaved Caspase-3 (Abcam, 1:500), anti-rabbit LC3 (Abcam, 1:3000), anti-rabbit p62 (Proteintech, 1:1000), anti-rabbit Beclin1 (Abcam, 1:1000), anti-rabbit ASK1 (Abcam, 1:500), rabbit anti-phospho-ASK1 (Thermo Fisher, 1:500), anti-mouse JNK (Proteintech, 1:3000), rabbit anti-phospho-JNK (Abcam, 1:1000), and anti-mouse β-actin (Proteintech, 1:2000). The mouse β-actin was used as internal reference protein. After being washed, membranes were then incubated with anti-rabbit IgG (H+L) (CST, 1:15000) or anti-mouse IgG (CST, 1:15000) fluorescent secondary antibodies. Densiometric scan and Image J were used to quantify the relative protein levels.