Ab1871

The ipsilateral brain tissues were removed 24 h after ICH and lysed in RIPA lysis buffer (Biocolors, 11,814,389,001, Shanghai, China). Protein extracts were electrophoretically resolved with SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Roche, GVWP02500, Basel, Switzerland). After blockade with 5% non-fat milk, the blots were then incubated with primary antibody against NLRP1 (1:2000, ABF22, Sigma, Missouri, USA), ASC (1:2000, SAB4501315, Sigma), pro-caspase-1 (1:2000, PRS3459, Sigma), caspase-1 (1:2000, AB1871, Sigma), or β-actin (1:2000, A1978, Sigma) overnight at 4 °C, followed by peroxidase-conjugated secondary antibody (Cell Signaling Technology, MA, USA). The blots were visualized using an electrochemiluminescence detection kit (Amersham, Little Chalfont, UK).

The colon tissue immunofluorescence staining procedures¹⁵ were performed as follows: the sections were fixed, washed and blocked using a 5% normal goat serum solution. Subsequently, primary antibodies were introduced, and the sections were incubated overnight at 4°C. Subsequently, the segments were washed and incubated with corresponding secondary antibodies (Cell Signalling Technology) for 1 h at room temperature. The specimens were counterstained with DAPI to facilitate nuclear visualization, acquiring images with a confocal microscope (Olympus). The study conducted an immunofluorescence analysis of NCM460 or HcoEpic cells.¹⁶ Briefly, NCM460 cells were fixed, permeabilized and blocked. Subsequently, the cells were incubated with the primary antibodies, anti‐LC3B (Abcam: ab192890), anti‐GSDMD (Abcam: ab219800), anti‐NLRP3 (Abcam: ab4207) and anti‐Caspase 1 (Sigma: AB1871), at 4°C overnight. Then, the cells were washed, incubated with the appropriate secondary antibodies and stained with DAPI to mark the nuclei. The samples were visualized using a confocal microscope (Carl Zeiss; Oberkochen).

After the mouse model was successfully prepared, midbrain was isolated and weighed, 1:10 (mass: volume ratio, 10 μl/1 mg of tissue) RIPA (Beyotime, China) protein lysis solution was added and lysed on ice for 40 min. After centrifugation at 16,000 × g for 15 min at 4°C, the supernatant contained whole cell protein. The protein supernatant was denatured in 95°C metal bath for 5 min by adding 5× loading buffer according to volume ratio and stored at −20°C. An equivalent amount of protein (60 μg) was electrophoresed on a polyvinylidene difluoride (PVDF) membrane using sodium dodecyl sulfate and polyacrylamide gels. The PVDF membrane was placed in 10% defatted milk powder‐TBST (pH 7.4, 10 mM TriS‐HCl, 150 mM NaCl, 0.1% Tween‐20), shaken, and sealed at room temperature for 1 h, then the primary antibody prepared by 5% BSA‐TBST was added, mouse anti‐NLRP3 (AG‐20B‐0014‐C100, Adipogen, 1:1000), Rabbit anti‐Caspase‐1 (AB1871, Millipore, 1:1000), and mouse anti‐β‐actin (AC026, ABclonal, 1:1000) overnight at 4°C. PVDF was washed for 10 min × 3 times, and ECL (Pierce) was added to develop the colors by Image Quant LAS 4000 Mini (GE). The gray value of target protein was compared to internal reference β‐actin in semiquantitative analysis (Image J).