Immunohistochemical studies were also conducted. The sections were incubated at 4°C with primary antibodies for 16 hours. The primary antibodies used in the study included (1) a polyclonal goat antibody to α1-PI (1:200; MP Biomedicals, Aurora, Ohio), (2) a polyclonal rabbit antibody to Sp1 (PEP 2, 1:100; Santa Cruz Biotechnology, Santa Cruz, California), (3) a polyclonal rabbit anti–MMP-1 antibody (1:100; Lab Vision, Fremont, California), (4) a monoclonal mouse antibody specific to MMP-2 (A-Gel VC2, 1:50; Lab Vision), and (5) a monoclonal mouse antibody to MMP-3 (SL-1 IID4, 1:20; Lab Vision). Before primary antibody incubation for MMPs, an antigen retrieval method was applied on each section with 8-mol/L buffered urea (pH 3.5) at room temperature for 2 hours. Biotinylated donkey anti–goat IgG (1:250; Jackson ImmunoResearch Laboratories, Inc, West Grove, Pennsylvania), donkey anti–rabbit IgG (1:250; Jackson ImmunoResearch Laboratories, Inc), and donkey anti–mouse IgG (1:250; Jackson ImmunoResearch Laboratories, Inc) were used as secondary antibodies at room temperature for 45 minutes. The color reaction for the anti-Sp1 was performed with fast red TR/naphthol AS-MX phosphate (Sigma-Aldrich, St Louis, Missouri) after sections were incubated with alkaline phosphatase conjugated Extravidin (1:50; Sigma-Aldrich). For α1-PI, MMP-1, MMP-2, and MMP-3, sections were incubated with avidin-biotin complex (Vectastain; Vector Laboratories, Burlingame, California) for 45 minutes followed by incubation with 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich). After mounting in Permount (α1-PI, MMP-1, MMP-2, and MMP-3) or aqueous mounting fluid (Sp1), the staining intensity in each experiment was scored by 3 masked observers (D.P.E., B.Y., H.N.) on a scale from 0 to 3, with 0 indicating no staining and 3 the most intense staining. Experiments were repeated twice.