Cox 2 antibody

Human Aβ_1–42 was purchased from EMD Millipore Corporation (Billerica, MA, USA). Ori was obtained from Chengdu Must Bio-Technology Company (Sichuan, China). Neurobasal media and B27 were bought from Life Technologies Corporation. MAP-2 antibody was purchased from Abcam (Cambridge, MA, USA). COX-2 antibody was bought from Santa Cruz Biotechnology (Santa Cruz, USA). PSD-95 antibody, synaptophysin antibody and CREB antibody were obtained from CST (Cell Signaling Technology, USA). 4', 6'-diamidino-2-phenylindole (DAPI), VDAC1, BDNF, p-TrkB, TrkB, p-CREB and β-actin antibodies were purchased from Bioworld Technology (Bioworld, USA). Lamin B1 antibody and HRP-conjugated secondary antibodies were also obtained from Bioworld.

Human monocytes were lysed using RIPA buffer supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). The lysates were separated by NuPAGE™ 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose membranes using the Trans-Blot^® Turbo™ Transfer System (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Membranes were blocked with 50% Odyssey Blocking Buffer in PBS-T (0.1% Tween20 in PBS) buffer and incubated with appropriate antibodies overnight at 4 °C. COX-2 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) or Cell Signaling Technologies (Danvers, MA, USA). Antibodies against mPTGES1 and vinculin were purchased from Millipore Sigma (Burlington, MA, USA). Primary antibody incubations were followed by incubation with IRDye^® secondary antibodies for 1 h at room temperature. Immunoblots were visualized and imaged using the Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Protein band intensity was measured using Image J software (National Institutes of Health, Bethesda, MD, USA) and normalized to vinculin.

The cell line—macrophage cell (RAW 264.7)—used for the measurement of cell viability was purchased from the Korean Cell Line Bank (Seoul, Korea). The reagents for cell culture, fetal bovine serum (FBS), and Dulbecco’s modified eagle medium (DMEM) were purchased from Sigma Aldrich Co., Ltd. (St. Louis, MO, USA). The reagents used for anti-inflammatory measurement experiments, MTT, Griess reagent, RIPA lysis and extraction buffer, LPS, protease inhibitor, phosphatase inhibitor, and nuclear and cytoplasmic extraction reagents were purchased from Sigma Aldrich Co., Ltd. The iNOS antibody, COX−2 antibody, donkey anti-mouse IgG-HRP, and mouse anti-rabbit IgG-HRP for the Western blot analysis were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). JNK antibody, p-JNK antibody, ERK1/2 antibody, p-ERK1/2 antibody, and NF-kB (p65) antibody were obtained from Cell Signaling Technology (Beverly, MA, USA).

The immunohistochemical staining procedure was used according to (Saber et al., 2019 (link)), for antigen retrieval, dewaxed sections were put in a citric acid buffer solution of 0.05 M, pH 6.8. The sections were then treated with 0.3% H₂O₂ and protein block. After that, incubated with Bax antibody (Santa Cruz, Cat: sc-7480, 1:100 dilution), caspase-3 polyclonal antibodies (Invitrogen, Cat: PA5-77887, dilution 1/100), p53 antibody (Santa Cruz, Cat: sc-126, 1:100 dilution), Bcl-2 antibody (Santa Cruz, Cat: sc-7382, 1:100 dilution), COX-2 antibody (Santa Cruz, Cat: sc-19999, 1:100 dilution), and Ki-67 (Santa Cruz, Cat: sc-23900, 1:100 dilution). Then for 30 min at 37°C with a secondary antibody linked to horseradish peroxidase. Following each process, phosphate buffer saline was applied to the slides three times. Sections were exposed for 3 min to the 3,3′-diaminobenzidine tetrahydrochloride reagent. Finally, slides were counterstained with Mayer’s hematoxylin, washed with distilled water, and mounted with DPX.
Using a digital camera attached to a microscope (Olympus CX21, Japan), slides were inspected under a microscope, and digital micrographs were taken. By using ImageJ software; version 1.54 D, Java 1.8.0_354, Positive expressions of staining intensity were measured and presented as a percentage of positive area per 6 high power fields.

OS-732 cells were purchased from Beijing Jishuitan Orthopaedic Laboratory. RPMI-1640 medium (Gibco Company), lipofectamine2000 transfection reagent (Invitrogen), Trizol (Sangon, Shanghai), one-step RT-PCR kit (TaKaRa, Dalian), Matrigel in vitro membrane matrix gel (Peking University), and Transwell cell culture membrane (Coring Costar) were used. Specific primers for COX-2, uPA and uPAR were synthesized by Sangon Shanghai, and COX-2 antibody was purchased from Santa Cruz. Our study was approved by an ethics committee of the Shanghai tenth People’s Hospital, China.