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; Laboratory Vision, Fremont, California), (4) a monoclonal mouse antibody specific to MMP-2 (A-Gel VC2, 1:50; Laboratory Vision), and (5) a monoclonal mouse antibody to MMP-3 (SL-1 IID4, 1:20; Laboratory Vision). Before primary antibody incubation for MMPs, an antigen retrieval method was applied on each section with 8M buffered urea (pH 3.5) at room temperature for 2 hours. Biotinylated donkey anti–goat IgG (1:250), donkey anti–rabbit IgG (1:250), and donkey anti–mouse IgG (1:250; all from Jackson ImmunoResearch, West Grove, Pennsylvania) were used as secondary antibodies at room temperature for 45 minutes. The color reaction for the anti-Sp1 was carried out with Fast Red TR/Naphthol AS-MX phosphate (Sigma, St Louis, Missouri) after sections were incubated with alkaline phosphatase–conjugated ExtrAvidin (1:50; Sigma). 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). After mounting in Permount (Thermo Fisher Scientific, Waltham, Massachusetts, for α1-PI, MMP-1, MMP-2, and MMP-3) or aqueous mounting fluid (Vectashield; Vector Laboratories, for Sp1), the staining intensity in each experiment was scored by 3 masked observers on a scale from 0 to 3, with 0 indicating no staining and 3 the most intense staining. Each experiment was repeated twice. Statistical analysis was performed using the Mann-Whitney U test to compare the staining intensity of keratoglobus and keratoconus corneas with those from normal controls. Within the corneas with a keratoglobus, immunostaining intensity was compared between the central and mid-peripheral areas. P < .05 was considered statistically significant.