Casp1

Fresh mouse brains were collected and sliced into 2 mm sections in a mouse brain mold (RWD Company; Shanghai, China). We chose the ipsilateral cerebral hemisphere (2 mm posterior to the bregma), including the cortex and striatum, for Western blotting. Proteins were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in the protein-free rapid blocking buffer (EpiZyme; Shanghai, China) for 15 min. Then, membranes were incubated with primary antibodies against NLRP3 (1:1000; Cell Signaling Technology), ASC (1:1000; Cell Signaling Technology), CASP1 (1:1000; Santa Cruz Biotechnology), β-actin (ACTB; 1:1000; Santa Cruz Biotechnology) for 16 h at 4 °C. Next, membranes were thrice washed with tris-buffered saline containing 0.1% Tween 20 (TBST) buffer for 15 min and incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) at room temperature for 1 h. The membranes were visualized using an enhanced chemiluminescence kit (ECL; Pierce; Waltham, MA, USA). Protein bands were quantified with the Image J. software.

The following antibodies were used in this study: HA tag (Santa Cruz Biotechnology, #sc-805, 1:1000 for western blot), GAPDH (Santa Cruz Biotechnology, #sc-47724, 1:1000 for western blot), ASC (Adipogen, #AL-177, 1:1,000 for western blot), CASP1 (Santa Cruz Biotechnology, #sc-622, 1:1000 for western blot), IL-1B (R&D systems, #AF-201, 0.1 µg/mL for western blot, 2 µg/mL for immunochemistry), FLAG (SigmaAldrich, #F3165, 1:2000 for western blot), GFP (Abcam, #ab290, 1:2,000 for western blot). IL-1B measurements were carried out with human IL-1B ELISA kit (BD, #557953, follow supplier instructions).

Brain sections were fixed in 4% paraformaldehyde (PFA) for 10 min, and the cell membrane was disrupted with 0.3% Triton X-100 for 10 min. Blocking was performed with 1% bovine serum albumin (BSA) for 1 h. Next, brain sections were incubated with antibodies against cluster of differentiation 11b (CD11b; 1:200, Cell Signaling Technology; Boston, MA, USA), CASP1 (1:200, Santa Cruz Biotechnology; Paso Robles, CA, USA), or ASC (1:200, Cell Signaling Technology) at 4 °C for 16 h. The sections were washed thrice with PBS and incubated at room temperature for 1 h with a secondary antibody corresponding to the primary antibody. The secondary antibody was washed off with PBS and stored at −20 °C after adding an antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies; Mulgrave, Australia). Deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed with a one-step TUNEL apoptosis assay kit (Meilunbio; Shanghai, China). Stained sections were observed with the TCS SP5 Confocal Scanning System (Leica; Wetzlar, Germany). Four fields along the peri-infarct regions were selected for each section.