Anti cox 2

Immunohistochemistry was performed using polyclonal antibody for SphK1 and COX-2. Tissue samples were recovered, fixed in 10% buffered formalin, and embedded in paraffin. Section (4 µm) were dewaxed and hydrated through graded ethanol, cooked in 25 mM citrate buffer, pH 6.0, in a pressure cooker for 10 min, transferred into boiling deionized water, and let to cool for 20 min. Tissue sections were then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity. The slides were incubated with anti-SphK1 (Abcam) and anti-COX-2 (Cell Signaling) antibodies at a working dilution of 1:200 overnight at 4 °C [18 (link)] and a secondary polymer-based detection system (EnVision+ System Labelled Polymer-horseradish peroxidase anti-rabbit; Dako). The chromogen was 3,3′diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA, USA) and sections were counterstained with haematoxylin. The specificity of technique was evaluated by negative controls (omitting the incubation with the primary antibody and incubating it with nonimmune sera). Areas of staining were analyzed by WinRoof version 6.3 (Mitani, Tokio, Japan) software with ten non-overlapping randomly chosen histological fields. Results were expressed as the percentage of stained cells in each field.

We use 10% SDS–PAGE gels to separated equal amounts of protein and transferred onto PVDF membranes for detection. The membranes were then sequentially incubated with specific antibodies, and the protein bands were detected using enhanced chemiluminescence. Anti-TFAP2B was purchased from Santa Cruz Biotechnology (sc-166441), anti-COX-2 was purchased from Cell Signaling Technology (#12282), and anti-GAPDH (10494-1-AP), anti-VEGF (19003-1-AP), anti-PEDF (26045-1-AP) were purchased from Proteintech (Wuhan, China).

SH-SY5Y cells and VM organotypic cultures were treated with a lysis buffer and protein concentrations were estimated by the Bio-Rad protein assay using bovine serum albumin as standard, as previously described [10 (link)]. Specific primary antibody anti-iNOS (BD Transduction 610329; 1/500), anti-COX2 (Cell Signaling #4842 1/500), anti-Bid (1/500; Santa Cruz Biotechnology), anti-Bad (1/500; Santa Cruz Biotechnology), anti-IκBα (Santa Cruz Biotechnology sc-371; 1/500), anti-NFκB p65 (Santa Cruz Biotechnology sc-372; 1/500), anti-Bcl2 (Santa Cruz Biotechnology sc-7382; 1/500) and anti-Bax (Santa Cruz Biotechnology sc-7480; 1/500) were mixed in 1× PBS, 5% w/v nonfat dried milk, 0.1% Tween-20 (PMT) and incubated at 4°C, overnight. Signals were detected as described above [11 (link)].

After the cell treatment, cells were rapidly washed with ice-cold phosphate buffered saline and scraped in lysis buffer for protein extraction (10 mM Tris/HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM Sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5% Triton X-100, 1 mM PMSF and protease inhibitor cocktail from Thermo Scientific). Lysed cells were centrifuged at 14.000 g for 20 min. SDS-PAGE and blotting procedure were carried on as previously described38 (link). Immunoblots were incubated with the appropriate antibody (anti-PGRN diluted 1:1000, Santa Cruz, CA, USA; anti-NOS2 diluted 1:1000, Cell Signalling, MA, USA; anti-COX2 diluted 1:50, anti-VCAM-1 diluted 1:1000, Cell Signalling, MA, USA; anti-MMP13 diluted 1:500, Santa Cruz, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore, MA) using anti-rabbit (GE Healthcare, UK) horseradish-peroxidise-labelled secondary antibody diluted 1:2000. To confirm equal loading in each sample, the membranes were stripped in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-GAPDH antibody diluted 1:30000 (Sigma, MO, USA). The images were captured and analyzed with an EC3 imaging system (UVP). Densitrometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).