Rabbit anti caspase 1

We used a freezing microtome (Thermo Scientific, Waltham, MA, USA) to obtain coronal sections (15 μm thickness) from the brain tissue with ischemic lesions. The slices were then permeated with 0.5% Triton X-100 for 20 min, blocked with 10% donkey serum at 37 °C for 1 h, and incubated overnight with primary antibodies at 4 °C. The primary antibodies were as follows: rabbit anti-NLRP3 (1:200, Bioworld Technology, Nanjing, China); rabbit anti-Caspase-1 (1:100, Affinity biosciences, Changzhou, China); and rabbit anti-IL-1β (1:100, Abcam, Waltham, MA, USA). The following day, brain slices were washed three times with phosphate-buffered saline (PBS) and incubated with secondary antibodies for 1 h at 37 °C. After three additional washes with PBS, they were treated with Fluoromount-G containing 4′,6-diamidino-2-phenylindole (DAPI; Southern Biotech, Birmingham, AL, USA) to stain the nuclei. Immunofluorescence was observed using a laser scanning confocal microscope (Zeiss LSM880, Oberkochen, Germany). ImageJ software was used to measure the number of positive cells in five different areas around the cerebral infarction [25 (link)].

The right brain cortex and retina protein samples (10 μg) were subjected to 8–12% SDS-PAGE and then transferred to PVDF membranes (Merk Millipore, Burlington, MA, USA). Blocked with 5% normal goat serum (NGS) for 2 h, the membranes were incubated with primary antibodies: rabbit Anti -NLRP3 (1:1000, Cat# ab263899; Abcam), rabbit anti-GSDMD (1:500, Cat# AF-4012; Affinity), rabbit anti-caspase-1 (1:1000, Cat# AF-4022; Affinity), and rabbit anti-β-actin (1:200; Cat# AF5003; Beyotime Biotechnology, Shanghai, China) at 4 °C for 24 h. The membranes were then incubated with anti-rabbit secondary antibodies (1:500; Cat# A0239; Beyotime Biotechnology, Shanghai, China) at 4 °C for 2 h. The protein signals were detected by Immobilon Western Chemiluminescent HRP substrate (Merk Millipore, Burlington, MA, USA) and analyzed using ImageJ analysis software (https://imagej.net/Fiji/Downloads, accessed on 30 May 2017, Bethesda, MD, USA).