0.2 μm polyvinylidene fluoride membrane

Total protein was extracted from the penumbral region of ischemic hemispheres, different conditions-treated ECs or BV₂ microglia as described previously (Wang et al., 2016 (link)). Then, 20 μg of protein samples were subjected to 10–15% Bis-Tris gel electrophoresis and transferred onto a 0.2 μm polyvinylidene fluoride membranes (Millipore Corporation, United States). The membranes were blocked with 5% non-fat dry milk-TBS-0.1% Tween 20 for 2 h and washed. Then, the membranes were incubated with primary antibodies against TLR4, MyD88, p-p65, p65 (1:200, all from Santa Cruz, CA, United States), PPARγ, NLRP3, caspase-1, lba1, CD68 (1:1000, both from Abcam, United States), occludin, ZO-1 and β-actin (1:1000, all from Cell Signaling Technology, United States), overnight at 4°C. This was followed by incubation with appropriate horseradish conjugated secondary antibodies for 2 h. Immunoreactivity was detected by the BeyoECL Plus kit (Beyotime, China) on an image system (ChmiScope 2850; Clinx Science Instruments, China); band intensities were analyzed and calculated.

MSCs were collected and homogenized in RIPA lysis buffer containing protease inhibitors (Solarbio, Shanghai, China), and a BCA Protein Assay Kit (Thermo Scientific) was used to quantify the total protein. The total proteins were diluted in 4× SDS-PAGE loading buffer, boiled for 10 min, electrophoresed on 10% polyacrylamide gels, and blotted on 0.2 μm polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After blocking with 5% nonfat milk for 2 hr at room temperature, the blots were incubated with primary antibodies overnight at 4°C followed by secondary antibodies at room temperature for 2 hr. An anti-6×His tag (β-GAL^H363A) antibody was used. Signals were generated by using enhanced chemiluminescence reagent (Millipore, USA) and were captured by using a Tanon-5200 Chemiluminescence Imaging System (Tanon Science & Technology Co Ltd., Shanghai, China). Quantification was performed using ImageJ software. Antibodies are listed in Supplementary file 1.

Host tissues from around metacestodes were lysed on ice using radioimmunoprecipitation assay buffer (Thermo Scientific) containing phenylmethylsulfonyl fluoride, and the lysates were centrifuged at 4 °C and 12,000 r.p.m. for 10 min. The supernatant was collected, diluted in 5× loading buffer and boiled in a 95 °C water bath for 10 min. The prepared protein samples were preserved at − 80 °C after measurement of the protein concentration. The protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis at 20 μg per lane and transferred to 0.2-μm polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany). Nonspecific antigens on the membrane were blocked with Tris-buffered saline 0.05%—Tween 20 containing 5% nonfat milk at room temperature for 1 h. The polyvinylidene fluoride membranes were subjected to immunoblotting with primary antibodies against MMP2 (1:1000; Abclonal, Wuhan, China), MMP9 (1:1000; Abclonal) and β-actin (1:1000; Abclonal) at 4 °C overnight. The next day, they were washed and incubated with horseradish peroxidase-labelled goat anti-rabbit IgG (1:5000) at room temperature for 1 h. After washing, an enhanced chemiluminescence (Thermo Scientific) method was used to expose the bands, and the greyscale values were normalized to that of β-actin.