Bs 10411r

Protein was extracted from glioma cells and measured through the BCA kit (Beyotime Biotechnology, China) [18 (link)]. Then, the protein was extracted using SDS-PAGE (10%) and transformed into PVDF membranes (Millipore, USA). Afterwards, membranes were incubated using 5% skimmed milk and incubated with primary antibodies under 4°C overnight. The antibodies are as follows: anti-Bax (1: 2, 000, bs-28034R, Bioss, China), anti-Bcl-2 (1: 2, 000, bs-4563R, Bioss, China), anti-MMP-2 (1: 2, 000, bs-20705R, Bioss, China), anti-MMP-9 (1: 2, 000, bs-22502R, Bioss, China), anti-Cleaved caspase-3 (1: 2, 000, bsm-33199M, Bioss, China), anti-Cleaved caspase-9 (1: 2, 000, bs-3082R, Bioss, China), anti-Cox-2 (1: 2, 000, bs-10411R, Bioss, China), and anti-β-actin (1: 2, 000, bs-0061R, Bioss, China) with β-actin being the endogenous control. Afterwards, membranes were incubated for 1 h using a secondary antibody (1: 2, 000, bs-0311P-HRP, Bioss). Finally, ECL (Millipore, USA) was utilized to observe protein blots and quantified using ImageJ software (version 4.3; National Institutes of Health).

The VEGF, CD34, TGF-β1, COX-2, TIMP-1 and MMP-2 expressions in peritoneal adhesive tissues were detected by immunohistochemical staining. The detailed procedures were conducted as previously described^{61 (link)}. The primary antibodies anti- CD34 antibody (bs-9752R, BIOSS, China, 1:300 dilution), anti- VEGF antibody (bs-0279R, BIOSS, China, 1:300 dilution), anti-TGF-β1 antibody (sc-146, Santa Cruz Biotechnology, USA, 1:400 dilution), anti-COX-2 antibody (bs-10411R, BIOSS, China, 1:300 dilution), anti-TIMP-1 antibody (bs-4600R, BIOSS, China, 1:300 dilution) and anti-MMP-2 antibody (bs-4599R, BIOSS, China, 1:300 dilution) were incubated overnight at 4 °C, and a secondary antibody was incubated for 1 h at room temperature. The staining was assessed in a blind manner using a light microscope, and a representative field was chosen for as application. The semi-quantitation of immunohistochemical staining was performed, and the intensity was scored as follows: 0, negative; 1, mild; 2, moderate; and 3, severe. The extent of staining was assessed based on the percentage of positive cells and was scored as follows: 0, negative; 1, 1–25%; 2, 26–50%; 3,51–75%; and 4,76–100%. The staining scores were the sum of the intensity and extent scores, and ranged from 0 to 7. The final staining score was represented as the mean in each group.