Ab2462

At 3 months, rats in all groups (n = 6 rats per group) were anesthetized by intraperitoneal as previous mentioned. Rats were sacrificed and perfused with phosphate‐buffered saline followed by perfusion with 4% paraformaldehyde dissolved in PBS. Then, the brain was removed and fixed in 4% paraformaldehyde for 24 h. Immunohistochemistry (IHC) was performed as previously described.²⁸ The primary antibodies for against the following proteins were used: P‐EGFR (Cell Signaling, 3777), MMP‐2 (Abcam, ab2462), and MMP‐9 (Abcam, ab86657), α‐SMA (Abcam, ab5694). All slides were observed at 20× or 40×, and all images were acquired using an Olympus DP80 microscope.

After rat brain was fixed in paraformaldehyde, the paraffin section was made as previously described.²⁵ Then, the paraffin section was baked for 15 min. Treat the sections with xylene and alcohol. Antigen retrieval: use citrate buffer solution (pH 6.0) repair sections for 10 min with high pressure, then natural cooling to room temperature. Wash sections by PBS for 3 times × 5 min. Transmembrane by heat: penetrate section by 3% TritionX‐100 for 30 min and wash section by PBS for 3 times for 5 min. Block section by 3% BSA for 30 min. Wash section by PBS for 3 times × 5 min. Incubate with primary antibody: dilute the primary antibody by antibody diluent and incubated overnight at 4°C. Wash section by PBS for 3 times × 5 min. Incubate with fluorescent second antibody: dilute antibody by PBS at 1:300 and incubate at 37°C for 30 min in dark. Wash section by PBS for 3 times × 5 min. Stain section by DAPI at room temperature for 2 min in dark. Wash section by PBS for 3 times × 5 min. Seal section by glycerol. Photographs were taken using Olympus DP80 fluorescence microscope. The primary antibodies for the following proteins were used: p‐EGFR (Cell Signaling, 3777), MMP‐2 (Abcam, ab2462), α‐SMA (Abcam, ab5694), and TNF‐α (Proteintech, 17590‐1‐AP). The fluorescent second antibodies (Invitrogen, A21206 and A21203) were used.

Proteins were extracted using RIPA lysis (Beyotime, Jiangsu, China) buffer and were collected. Then, protein concentration was determined using a BCA assay kit (Thermo Scientific, Waltham, MA, USA). The protein samples were separated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene fluoride (PVDF, EMD Millipore, Billerica, MA, USA) membranes. Membranes were blocked with 5% skim milk for 1 hour and then incubated with primary antibodies against CDK4 (ab199728, 1 : 2000, Abcam), Cyclin D1 (ab226977, 1 : 500, Abcam), p53 (ab131442, 1 : 1000, Abcam), p21 (ab7960, 1 : 2000, Abcam), MMP2 (ab2462, 1 : 5000, Abcam), MMP9 (ab76003, 1 : 2000, Abcam), E-cadherin (ab18203, 1 : 1000, Abcam), N-cadherin (ab18203, 1 : 1000, Abcam), cleaved caspase-3 (ab2302,1 : 1000, Abcam), cleaved PARP (ab32064, 1 : 1000, Abcam), Bcl-2 (ab32124, 1 : 1000, Abcam), Bax (ab7977, 1 : 1000, Abcam), CD86 (ab7977, 1 : 1000, Abcam), CD206 (ab7977, 1 : 1000, Abcam), iNOS (ab7977, 1 : 1000, Abcam), Arg-1 (ab7977, 1 : 1000, Abcam), and GAPDH (ab8245, 1 : 1000, Abcam) at 4°C for overnight. Subsequently, the membranes were probed with horseradish peroxidase- (HRP-) conjugated secondary antibodies (Abcam) at room temperature for 1 hour. The band was visualized using an enhanced chemiluminescence technique (Thermo Fisher Scientific).

SP method was used for immunohistochemistry according to the manufacture’s protocol. After incubation with 1% H₂O₂ in methanol and 10% goat serum for 30 min, the primary antibodies to MMP2 (ab2462, Abcam, Hong Kong, China), MMP9 (ab58803, Abcam, Hongkong, China), and monoclonal mouse anti-rabbit macrophage (Clone RAM11, Dako, Denmark) were incubated overnight at 4 °C. Sections were incubated with biotinylated anti-mouse second antibody for 20 min, and visualized with diaminobenzidine tetrahydrochloride. Immunofluorescence was used to detect the assembly of smooth muscle cells (SMCs). Sections were incubated overnight with mouse monoclonal anti-alpha-smooth muscle actin (A2547, Sigma-Aldrich (Shanghai) Trading Co., Ltd, China), and subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse IgG. Semiquantitative analyses for SMCs content was calculated as the mean area of 6 fields per rabbit.