Anti caspase 1

Western blotting was performed as described previously (25 (link)). The cytosolic and nuclear fraction preparation was performed using ExKine™ Nuclear and Cytoplasmic Protein Extraction Kit (Abbkine, China) according to the manufacturer’s instructions. The primary antibodies used were as follows: anti-NLRP3 (1:1,000; Cell Signaling, Danvers, MA, USA), anti-XIAP (1:1,000; Enzo Life Sciences, Farmingdale, NY, USA), anti-ASC (1:1,000; Abbkine), anti-caspase-1 (1:1,000; Novus Biologicals), anti-TLR4 (1:1,000; Bioworld Technology), anti-myeloid differentiation factor88 (MyD88, 1:1,000; Cell Signaling), anti-NF-κB (1:1,000; Cell Signaling), anti-Lamin B1 (1:1,000; Cell Signaling), anti-β-tubulin (1:1,000; Abbkine), and anti-β-actin (1:5,000; Enzo Life Sciences). The membranes were then washed thrice with Tris-buffered saline with Tween-20 (TBST) and incubated with secondary antibodies conjugated with horseradish peroxidase. Immunopositive bands were enhanced with chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA) and visualized using the Fusion Solo X (Vilber Lourmat, Collegien, France).

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).

NLRP3 subunit and caspase-1 were immunostained using monoclonal anti-NLRP3 (R&D Systems, Minneapolis, MN, USA) and anti-caspase-1 (for activated caspase-1; Novus Biologicals, Centennial, CO, USA) at a 1:500 dilution.

Protein expression of NLRP3 and caspase-1 was determined using protein analysis of thoracic aortas of in vivo and in vitro all groups. Samples were homogenized in lysis buffer and proteins were collected. Proteins (30 μg) were separated by electrophoresis on 10 or 12% polyacrylamide gels, transferred to 0.22 μm nitrocellulose membranes and blocked using 5% bovine serum albumin (BSA) in Tris buffered saline (TBS) and 0.1% Tween 20 for 1 h. Primary antibodies were incubated overnight at 4°C as follows: anti-NLRP3 (1:500 dilution; R&D Systems), anti-caspase-1 (1:1,000 dilution; Novus Biologicals), anti-β-actin-peroxidase (1:5,000 dilution; Sigma-Aldrich).