, Solon, OH) in TBST for 1 hour, and incubated overnight with primary antibodies, including anti-collagen I, III and IV (Southern Depsipeptide order Biotech, Birmingham, AL), anti-laminin (Sigma-Aldrich),

anti-decorin (Abcam Inc., Cambridge, MA), anti-fibronectin and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by appropriate horseradish peroxidase–conjugated secondary antibodies. Wet membranes were incubated with Pierce ECL Western blotting substrate (Thermo Scientific, Rockford, IL) for 1 minute and scanned in a Fujicolor LAS-3000 system (Fujifilm USA, Inc.). As positive control for protein expression, protein standards containing collagen I and III (BD Biosciences, San Jose, CA), collagen IV, fibronectin and decorin (Sigma-Aldrich) at known concentrations were loaded simultaneously on the gel. Immunohistochemical analyses were performed in formalin fixed and paraffin embedded bioscaffold and human liver sections

using the same antibodies indicated above. Decellularized ferret liver matrices were dried by lyophylization and divided into segments of 4-6 mg by mass (n = 3 per time point). The segments were placed in 37°C PBS. Controls were maintained in PBS, whereas experimental samples were placed in 1 mg/mL (250 U/mL) collagenase II (Worthington Biochemical NVP-BEZ235 Co., Lakewood, NJ). Samples were then collected and dried again by lyophylization. The masses of the bioscaffold segments in the control and experimental samples were measured at 3,

6, 12, 24, and 48 hours of exposure collagenase and mass average and standard deviation were calculated for each time point. Fluoroscopy was carried out in a decellularized pig liver using a Siemens SIREMOBIL Compact L C-arm. Conray Iothalamate Meglumine (Mallinckrodt Inc., St Louis, MO) contrast agent was diluted at a ratio of 1:50 in distilled water and perfused through the vasculature at a rate of 30 mL/minute. Fluorescent microscope imaging of the capillary tree (n = 3) was obtained by perfusing 100 μg/mL fluorescein bound to 250 kDa dextran (Sigma-Aldrich) into the mouse liver bioscaffold. Dextran bound fluorescein was used so as to minimize diffusion of the fluorescein outside of the vasculature. A fluorescent light microscope was used to obtain the other single plane images. For three-dimensional reconstruction of the vascular MCE公司 network, sequential pictures were taken of the bioscaffold after injection of dextran bound fluorescein particles with a Nikon 600N confocal microscope (Nikon Inc., Melville, NY). The pictures were rendered for three-dimensional reconstruction with the software Volocity (Improvision Inc., Waltham, MA). The right lobe of the acellular ferret liver bioscaffold was sterilized using a gamma irradiator (J. L. Shepherd and Associates, Inc., San Fernando, CA) to provide a dose of 1.5 Mrad. Prior to transplantation, 150 U of heparin sodium (Abbott Laboratories, Inc., Abbott Park, IL) was injected into the bioscaffold using its portal catheter.