Sc 5538

In addition to histomorphological evaluation, serial sections were stained for COL2A1, NF-κB p65, MMP-13 and TIMP-1. After deparaffinization and rehydration of the tissue sections, the proteins were immunostained using a two-step method following the kit manufacturer’s instructions. The sections were incubated with rabbit polyclonal anti-COL2A1 antibody (sc-28887, 1:50; Santa Cruz), mouse monoclonal anti-NF-κB p65 antibody (sc-8008, 1:50; Santa Cruz), goat polyclonal anti-MMP-13 antibody (sc-31813, 1:50; Santa Cruz), or rabbit polyclonal anti-TIMP-1 antibody (sc-5538, 1:50; Santa Cruz) overnight at 4°C. The slides were washed three times in PBS followed by a 20 min incubation at 37°C with an anti-mouse/rabbit IgG detection system (PV-9000, Zhongshan Goldenbridge Biotechnology Co., China), anti-goat IgG detection system (PV-9003, Zhongshan Goldenbridge Biotechnology Co., Beijing, China) and visualized with diaminobenzidine (DAB). Nuclei were counterstained with hematoxylin for 5 min. Negative control sections were prepared with the same protocol as above, but the primary antibody was replaced by PBS. The optical density of stained slides was measured using image analysis software (NikonH600L Microscope and image analysis system, Japan). COL2A1 was expressed by relative intensity. NF-κB (p65), MMP-13 and TIMP-1 were expressed by percentage of positive cells.

Proteins were extracted from the left lobe using RIPA buffer containing protease inhibitor cocktail (Selleck, Houston, USA), and an equal amount of proteins (70 μg) from each sample was separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, USA). The membranes were blocked with Tris-buffered saline and Tween 20 buffer containing 5% fat-free milk powder for 1 h at room temperature and then incubated overnight at 4°C with different primary antibodies, including rabbit monoclonal to alpha smooth muscle Actin (α-SMA; 1:1000; ab32575; Abcam), rabbit polyclonal to TIMP-1 (1:100; sc-5538; Santa Cruz Biotechnology) and Monoclonal Mouse Anti-glyceraldehyde-3-phosphate Dehydrogenase (GAPDH; 1:10000; KC-5G4; Aksomics, Shanghai, China). After extensive wash, membranes were incubated with fluorescein-conjugated secondary antibody for 1 h (1:10000; LI-COR Biosciences, Lincoln, USA). Protein bands of interest were analyzed using the Odyssey Infrared Imaging System (LI-COR). GAPDH was used as the loading control.