Mouse anti human cd147

Samples of formalin-fixed lung tissue were embedded in paraffin, sectioned (4 μm) and stained with hematoxylin and eosin (HE) for morphometric measurement or with Alcian blue-periodic acid Schiff stain to assess the mucin glycoproteins in the epithelium. The tissue sections were deparaffinized and stained with mouse anti-human CD147 (1:100, Abcam) and mouse anti-human MUC5AC (1:200, Abcam) primary antibodies overnight at 4 °C; then, the sections were incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:1000, Abcam), and the color was developed using diaminobenzidine. In the negative controls, the primary antibody was substituted with phosphate-buffered saline (PBS). Immunochemistry staining of the lung tissue sections was evaluated at 200-fold magnification in four random fields per section. The mean staining intensity values of CD147 and MUC5AC in the airway epithelium were analyzed using Image-Pro Plus 7.0 software.

Cells were lysed in RIPA lysis buffer with protease and phosphatase inhibitors (Roche). Equal amounts of cell lysates from the protein samples were resolved by 10 and 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. These membranes were incubated with 5% skimmed milk or 5% BSA in PBST at room temperature for 1 h; after rinsing 3 times, the membranes were incubated overnight at 4 °C with the following specific primary antibodies: mouse anti-human CD147 (at 1:1000, Abcam), rabbit anti-human MMP9, rabbit anti-human p38 MAPK and rabbit anti-human phosphorylated p38 MAPK (at 1:1000, Cell Signaling Technology Company). This step was then followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:5000, Jackson) for 1 h at room temperature. The blots were visualized with an enhanced chemiluminescence kit (ECL plus). The intensity of each band was measured by using Quantity One software. The relative protein expression levels were determined by normalization to that of β-actin.