| Vitreous collagen extraction|
Collagen was extracted from the rabbit vitreous, using a modification of the Seery and Davison method . A total vitreous was mixed with 1 ml protease inhibitor cocktail (0.5 mg/l leupeptin, 1 mM Na2EDTA, 0.7 mg/l pepstatin, and 0.2 mM phenylmethylsulfonyl fluoride; Boehringer Mannheim, Indianapolis, IN), and homogenized at 0 °C in a Ten Broeck tissue grinder (Corning Glass Works, Corning, NY). The homogenate was centrifuged (100,000x g, 45 min) for 45 min at 4 °C in an ultracentrifuge (L3-50, Beckman, Palo Alto, CA). The insoluble residue, which included the collagen, was resuspended in 0.5 M acetic acid, treated with pepsin (10 mg of enzyme per 100 mg sample; pepsin, twice crystallized, Sigma, St. Louis, MO), and agitated overnight at 4 °C. After the enzyme treatment and centrifugation (11,000x g for 30 min), the pH of the supernatant was adjusted to 7.2 by the addition of 1 M NaOH, which then was dialyzed against 0.01 M Na2HPO4 at 4 °C overnight, dialyzed against distilled water at 4 °C overnight, and freeze-dried to yield solubilized vitreous collagen.