Other rats were injected with 2.0 mg/mL or 0.2 mg/mL rPAI-1 or vehicle at 14/2.5 and were killed 24 hours later. After retinal dissection, two retinas from each treatment were pooled and homogenized in 150 μL of extraction buffer (40 mM Tris-HCl, 110 mM Tris base (pH. 7.4), 150 mM NaCl, 5 mM CaCl, 5 mM MgCl2, and 1% Triton X-100) before flash-freezing. Samples were thawed and centrifuged at 20,800g for 8 minutes at 4°C. Protein concentration was measured in all samples with the BCA protein assay kit (Pierce), and an equivalent volume of each was affinity purified with an 8:1 ratio of sample volume to gelatin Sepharose 4B beads (Amersham Pharmacia Biotech, Piscataway, NJ) by incubation at 4°C with rocking for 1 hour. Samples were eluted in 30 μL of 2× Bio-rad zymogram sample buffer (Richmond, CA) plus 10% dimethyl sulfoxide (DMSO). A 20-μL aliquot of each sample was loaded on a 10-well, 10% gelatin zymography gel (Ready Gel; Bio-Rad) with appropriate markers and controls. The gel was run for 90 minutes at 100 V in 1× Tris-glycine-SDS buffer (20 mM Tris base, 200 mM glycine, 3 mM SDS). After incubation with shaking at 25°C in 1× zymogram renaturation buffer (Bio-Rad; 2.5% Triton X-100) for 45 minutes, the gel was left overnight (16–20 hours, optimally) at 37°C in 1× zymogram development buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35; Bio-Rad). The gel was stained for 20 minutes in Coomassie blue stain (0.5% Coomassie blue R-250, 40% methanol, 10% acetic acid in distilled water), rinsed briefly in distilled water, and then destained for up to 2 hours in 40% methanol plus 10% acetic acid. Zones of clearing that corresponded to the presence of proteinases in the gel were quantified using image-analysis software (Enhance; MicroFrontier, Des Moines, IA). The data are expressed as pixels per microgram protein.