Gelatinase activity in the tear fluid was measured by gelatin
zymography. Diluted tear samples (10 μl of tears diluted 1:11) were
incubated with sodium dodecyl sulfate (SDS)–gel sample buffer for 30
minutes at room temperature and analyzed by electrophoresis on a
SDS–polyacrylamide gel (10%) containing gelatin (1 mg/ml). After
electrophoresis, the proteins were renatured by removing SDS from the
gel using two washes of 0.25% Triton X-100 (30 min/wash). This was
followed by an 18-hour incubation at 37°C in the digestion buffer
consisting of 50 mM Tris–HCl (pH 7.4) containing 0.15 M NaCl, 10 mM
CaCl2, 2 μM ZnSO4, 1 mM
phenylmethylsulfonyl fluoride, 0.005% Brij 35, and 0.02%
sodium azide. After this incubation, the gel was briefly rinsed in
distilled water and stained with 0.25% Coomassie brilliant blue R250
prepared in 40% isopropanol solution for 1 hour. The gel was destained
with 7% acetic acid. Gelatinase activity in the gel was visible as a
clear area in the blue background, indicating an area where the gelatin
had been digested. The minimum sensitivity of this technique for
detecting gelatinase B is 0.05 ng/lane. The molecular weight of
gelatinases in the tear fluid was determined from molecular weight
standards (prestained broad range standards; Bio-Rad, Hercules, CA) and
purified rabbit 92-kDa pro–gelatinase B (0.1 ng; Oncogene Research,
Cambridge, MA) that were run in separate lanes on the gel. These gels
were photographed with a Polaroid camera, and the photographs were
scanned with an HP ScanJet 4C scanner (Hewlett-Packard, Palo Alto, CA).
The optical densities of bands in the digitized images were determined
with the Gel-Pro Analyzer gel analysis software program (Media
Cybernetics, Silver Spring, MD). The ratio of the optical density of
the 92-kDa gelatinase band in each tear fluid sample to the optical
density of the pro–gelatinase B positive control band (0.1 ng) was
used for statistical analysis.