Gelatin zymography

Excised livers from sacrificed mice, previously stored at −80 °C were used. Liver tissue samples (50 mg) were weighed and homogenized in saline (0.9% NaCl solution) in a ratio of 1:4 w/v. The samples were centrifuged at 6,500 rpm and 4 °C then the supernatant which contained the matrix metalloproteinases (MMPs) was collected. Specific MMP-2 and MMP-9 activities were detected by gelatin zymography¹⁸ performed on premade 10% polyacrylamide gels containing 0.1% gelatin according to the instruction provided by manufacturer (Invitrogen, USA). On each gel, recombinant MMP-2 and MMP-9 (Merck Millipore, USA) were loaded as standards according to the manufacturer’s specifications. The bands were visualized by staining for 30 min with a solution containing 0.1% Coomassie R-250 (Sigma Aldrich, USA) in 40% ethanol and 10% acetic acid, followed by destaining for 2 h at room temperature in a solution containing 10% ethanol and 7.5% acetic acid. The relative activities of MMP-2 and MMP-9 for each sample were quantified by NIH ImageJ 1.42q software.