Rabbit anti opn

PureLink DNAse set and PureLink RNA mini kit were purchased from Invitrogen (Grand Island, NY, USA). High-Capacity cDNA Reverse Transcription kit and Power SYBR green Mastermix were purchased from Applied Biosystems (Foster City, CA, USA). Polyvinylidene difluoride (PVDF) membranes were purchased from Bio-Rad (Berkeley, CA, USA). Amersham ECL Plus Western Blotting Detection reagents were purchased from GE Healthcare (Rockford, IL, USA). Xuesaitong injection (containing 400 mg PNS per ampule) was obtained from the Kunming Pharmaceutical Group Co., Ltd. (Yun Nan province, China). Rabbit anti-Notch3, mouse anti-SM22α, and rabbit anti-OPN were purchased from Abcam (Cambridge, MA, USA). TRITR-phalloidin, FITC-phalloidin, and mouse anti-α-SM-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-β-actin and mouse anti-β-tubulin were purchased from Sigma (St. Louis, MO, USA).

Double immunofluorescent staining was performed using the following primary antibodies: mouse anti-NF-κB p65 (dilution ratio: 1:150; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CTSK (1:150; Abcam), rabbit anti-RANKL (1:150; Proteintech Group Inc., Rosemont, IL, USA), rabbit anti-OPG (1:150; Abcam), rabbit anti-OPN (1:200; Abcam), and rabbit anti-OCN (1:200; Abcam). The sections were first blocked using 5% bovine serum albumin and were then incubated with a specific primary antibody, followed by species-matched secondary antibodies (Donkey Anti-Rabbit IgG H&L Alexa Fluor 594 and Donkey Anti-Rabbit IgG H&L Alexa Fluor 488; Abcam), at a 1:200 dilution. All tissue sections were subjected to autofluorescence quenching (Vector TrueVIEW; Vector Laboratories, Inc.) and mounted with VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc.) following the manufacturer’s protocol. Fluorescent images were acquired using a Leica TCS-SP8 confocal laser scanning microscope (Leica Biosystems, Wetzlar, Germany) within 48 h after mounting. The immunofluorescence expression of each sample was evaluated using the MFI; a.u.).

Proteins were removed from rat aortic SMCs following lysis and after intervention. The cells were divided into four groups, as described, and 40 µg of protein per lane was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Epizyme, Beijing, China), transferred to a polyvinylidene difluoride membrane (Millipore Sigma, Billerica, MA, USA), and blocked with western quick-blocking buffer (Beyotime) for 15 min at room temperature. The blocked membranes were then incubated overnight with primary antibodies, namely goat anti-SM22α (1:500; Abcam, Cambridge, UK), rabbit anti-OPN (1:1000; Abcam), and rabbit anti-MMP9 (1:1000; Abcam). After 12 h to 14 h, the membranes were incubated with a horseradish-peroxidase-conjugated secondary antibody (HuaBio, Hangzhou, China) for 1 h at room temperature. The immunoblots were probed using an enhanced chemiluminescence substrate (Thermo Fisher Scientific), and an imaging system (Bio-Rad Laboratories, Hercules, CA, USA) was used for blot detection and recoding of chemiluminescence. The results were normalized to that of GAPDH, and experiments were performed in triplicate.