Anti atg5

Cells were harvested, washed, and then lysed in the RIPA buffer (Beyotime, Shanghai, China) containing a cocktail of protease inhibitors (MCE, NJ, USA). After quantifying the protein concentration using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China), the cell lysates (30 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 10% non-fat dry milk in Tris-Buffered Saline and Tween-20 (TBST) for 1 h and incubated overnight with primary antibodies at 4 °C. After being washed three times. The bound antibodies were reacted with a secondary antibody. They were then visualized with a fluorescence imaging system (Sagecreation, Beijing, China) using enhanced chemiluminescence (Advansta, CA, USA). The primary antibodies included anti- β-Actin (20536-1-AP), anti-GPX4 (14432-1-AP), anti-p62 (18420-1-AP), anti-SLC7A11 (26864-1-AP), anti-DDIT4 (10086-1-AP), anti-LC3 (Proteintech, 14600-1-AP), and anti-ATG5 (Zen-bio, R23497).

The subventricular zone (SVZ) tissue was separated and homogenized to collect the protein samples at 3 days after hemorrhage. Equal amounts of protein samples (20 μg) were loaded on SDS-PAGE gels, electrophoresed, and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked and incubated overnight at 4°C with the following primary antibodies: anti-ZO-1 (1 : 1000; Abcam, USA), anti-NLRP3 (1 : 1000; Abcam, USA), anti-Caspase1 (1 : 1000; NOVUS, USA), anti-IL1beta (1 : 1000; GeneTex, USA), anti-Atg5 (1 : 1000; ZEN-BIO, China), anti-LC3B (1 : 1000; ZEN-BIO, China), anti-p62 (1 : 1000; ZEN-BIO, China), anti-pAMPK (1 : 1000; CST, USA), anti-AMPK (1 : 1000; CST, USA), anti-ULK1 (1 : 1000; CST, USA), anti-Beclin-1 (1 : 1000; CST, USA), and anti-β-actin (1 : 1000; CST, USA). Appropriate secondary antibodies (1 : 3000, CST; 1 : 5000, abcam) were selected to incubate with the membrane for 1 h at room temperature. The bands were probed with an ECL Plus chemiluminescence regent Kit (ThermoFisher, USA) and visualized with the image system (Bio-Rad, USA). Relative density of the protein immunoblot images was analyzed by Image J software (NIH, USA).

Under deep anesthesia, rats were sacrificed by transcardial perfusion with 100 ml normal saline followed by 50 ml 4% neutral buffered paraformaldehyde. Brains were fixed in 4% neutral buffered paraformaldehyde for 24 h at 4°C followed by 25% and 30% sucrose solution until brains were dehydrated fully. Then, brains were cut into 10 μm thick coronal sections using a cryostat (LM3050S, Leica, Germany) after being frozen at -80°C. Slides were washed with 0.01 M of PBS 3 times for 10 min and then incubated in 0.3% Triton X-100 for 30 min at room temperature. After being blocked with 5% BSA for 1 h at room temperature, the sections were incubated with primary antibody at 4°C overnight as follows: anti-CD31 (1: 200; Abcam, USA), anti-ZO-1 (1 : 200; Abcam, USA), anti-Caspase1 (1 : 200; NOVUS; USA), anti-IL1beta (1 : 400; GeneTex, USA), anti-Iba1 (1 : 200; Genetex, USA), anti-CD68 (1 : 200; Abcam, USA), anti-NLRP3 (1 : 200; Abcam, USA), anti-Atg5 (1 : 100; ZEN-BIO; China), anti-p62 (1 : 100; ZEN-BIO; China), and anti-NeuN (1 : 200; Abcam, USA). Then, the sections were washed with 0.01 M PBS and incubated with appropriate fluorescence-conjugated secondary antibodies (1 : 400; Invitrogen, USA) for 2 h at room temperature. The slides were observed and photographed under a fluorescence microscope (LSM880; ZEISS, Germany).