Following elastin immunostaining or picrosirius red staining, the entire liver section of each blinded slide was sequentially scanned either at ×100 (mouse) or ×80 (rat) magnification. Stained areas were quantified by Adobe Photoshop CS2 and expressed as percentage of total pixels. Immunoprecipitation was performed at 4°C using ice-cold buffers. Tissues were homogenized in lysis buffer (20 mM Tris HCl pH 8, 137 mM NaCl, 10% glycerol, 1% Triton X-100). Protein concentration was determined by Bradford Assay and equal amounts (500 μg) were diluted in 500 μL intraperitoneal lysis buffer and mixed with either anti-MMP-12 (Abcam) or anti

Selleck FDA approved Drug Library TIMP-1 (ClonTech) at a final concentration of 1 μg/mL and rotated overnight. Then 100 μL of MagnaBind goat antirabbit IgG (Thermo, UK) or antimouse IgG1 Magnetic Particles – DM BD IMag (BD Biosciences) were added and rotated for 1 hour. Beads were magnetically separated for 8 minutes and supernatants were kept aside and equal volumes (20 μL) were used in western blot analysis for GAPDH to confirm initial equal protein amounts.

Separated beads were washed 2 × 5 minutes in intraperitoneal lysis buffer. Samples were then resuspended in 25 μL buy Autophagy inhibitor zymography sample buffer (62.5 mM Tris-HCl, pH 6.8, 4% SDS, 25% glycerol, 0.01% Bromophenol Blue). Casein zymography was performed according to Poppelmann et al.26 with minor modifications. In short, samples

were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel containing 0.25% w/v skimmed milk powder. Following electrophoresis, gels were rinsed in deionized water and renatured in 2.5% Triton X-100 for 4 hours before incubation MCE公司 in activity buffer (50 mM Tris, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35, pH 7.5) at 37°C for 72 hours. Subsequently, the gel was stained in SimplyBlue SafeStain (Invitrogen) before destaining in water. Proteolytic activity was detected as destained bands against a background of Coomassie-stained casein. All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS software. To ensure normality and equality of variances, the data were log-transformed prior to analysis. Following transformation the groups were compared using the test indicated in each figure legend. As we have previously described, administration of CCl4 to rats for either 4 or 8 weeks leads to reversible fibrosis and early cirrhosis, respectively, whereas 12 weeks administration leads to micronodular cirrhosis.4 Picrosirius red staining of rat liver tissue after CCl4 administration showed increasing accumulation of collagen, detectable following 4, 8, and 12 weeks of injury (Fig. 1A1-4). Histomorphometric analysis (Fig.