Ab124131

Ipsilateral basal cortical samples facing blood clots were extracted. Western blotting was performed as described previously (25 (link)). Cortical samples were homogenized and centrifuged (1,000 × g, 10 min, 4°C). The supernatant was further centrifuged, and then, the protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). An equal amount of protein (50 μg) was suspended in loading buffer, denatured at 95°C for 5 min, and loaded on an SDS-PAGE gel. After being electrophoresed and transferred into polyvinylidene fluoride membranes, the membrane was blocked with nonfat dry milk buffer for 2 h and then incubated overnight at 4°C with the primary antibody for AIM2 (ab93015, 1:1,000, Abcam), pannexin-1 (ab124131, 1:1,000, Abcam), ASC (ab155970, 1:1,000, Abcam), caspase-1 (ab1872, 1:1,000, Abcam), P2X7R (1: 1,000, APR-004, Alomone, Jerusalem), IL-1β (SC-23460, 1:500; Santa Cruz), IL-18 (ab71495, 1:1,000; Abcam), and GAPDH (1:10,000, Fitzgerald, 10R-G109A). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein band densities were detected by x-ray film and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry staining was conducted as previously described [9 (link)]. In brief, after anesthetizing the mice with isoflurane, the animals were perfused with saline followed by 4% PFA. The sciatic nerves and the DRG were removed and post-fixed in 4% PFA for 8–12 h. The sciatic nerve sections and DRG slices (12 μm) were sectioned using a cryostat machine followed by immunohistochemistry staining. After washing, the slices were permeabilized with 0.3% Triton X-100 for 10 min, and blocked with 10% BSA for 2 h at room temperature, and processed for immunostaining with the following primary antibodies: S100 β (1:400, mouse; Sigma, AMAB91038), NF200 (1:300, mouse; Sigma, N0142), Panx1 (1:200, rabbit; Abcam, ab124131) overnight at 4 °C. Then, the slices were incubated with a mixture of cy3 and 488-, or cy3 and cy5-conjugated secondary antibodies (1:1000; Jackson Immuno Research) for 2 h at room temperature. For the staining of neurons in the DRG slices, Nissl staining (1:200, Thermo, N-21483) was applied and performed via the manufacturer’s instructions. Stained samples were examined using a fluorescence microscope (Nikon), and images were captured with a CCD Spot camera.

Nuclear and cytosolic fractions of the spinal cords of mice were prepared at six time points. Membrane and cytosolic fractions of cultured astrocytes were also prepared by the same procedure. A total of 20 μg protein lysates was then resolved by 10% SDS-PAGE, transferred to a PVDF membrane, and probed with rabbit monoclonal antibodies against SGK-1 (1:1,000; ab32374, Abcam, Cambridge, UK), GCR (1:1,000; sc1002, Santa Cruz Biotechnology, Santa Cruz, CA), Pannexin-1 (1:1,000; ab124131, Abcam), Actin (1:1,000; sc1616-HRP, Santa Cruz Biotechnology) or POL2 (1:1,000; sc900, Santa Cruz Biotechnology). The specificities of antibodies for their target proteins have been validated in previous studies58 (link)59 (link)60 (link). Specific antigen–antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies and a chemiluminescence reagent (Nacalai Tesque, Kyoto, Japan).