Goat anti rabbit immunoglobulin g

Formalin-fixed and paraffin-embedded tissues were cut into 4-mm thick sections and subjected to immunohistochemistry using En Vision staining, following the manufacturer’s instructions. The sections were initially blocked with 5% BSA and then incubated with specific primary antibodies, including anti-TIMP-2 antibody (1:50, Santa Cruz, CA, USA), anti-MMP-2 antibody (1:200, Abcam Inc, MA, UK), or anti-TGF-β1 antibody (1:150, Boshide Biotech, Wuhan, China). The incubation was performed overnight at 4°C. Subsequently, the sections were incubated with a secondary antibody, goat anti-rabbit immunoglobulin G (Santa Cruz, CA, USA). As a negative control for staining, some tissue sections were incubated with phosphate-buffered saline (PBS) without primary antibodies. For image analysis, the Optical Density (OD) was calculated using Image-Pro Plus 6.0 image processing software based on five randomly chosen microscopic fields.

Forty-eight hours after miRNA mimics or miRNA inhibitor transfection, total proteins were isolated from tissues and cell lines with radioimmunoprecipitation (RIPA) lysis buffer (P0013B, Beyotime, People’s Republic of China). The supernatants containing the whole protein extracts were obtained after centrifugation of the lysates at 12,000× g for 20 minutes at 4°C. The protein concentrations were determined by enhanced bicinchoninic acid protein assay kit (Beyotime). Heat-denatured protein samples (50 µg per lane) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel and transferred to an Immobilon-P transfer membrane (EMD Millipore). The membrane was blocked in 5% non-fat dry milk in Tris-buffered saline (pH 7.4) containing 0.05% Tween-20 to block nonspecific binding, followed by incubation overnight at 4°C with a primary rabbit polyclonal antibody against human MMP16 (1:200, Boster, People’s Republic of China), MMP2 (1:250, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and was blotted with goat anti-rabbit immunoglobulin G (1:3,000, Santa Cruz Biotechnology Inc., USA). Then GAPDH was used as a loading control. Signals were detected by secondary antibodies labeled with HRP, and the bound antibody was detected with the use of enhanced chemiluminescence detection reagents (Beyotime) according to the manufacturer’s instructions.