Mmp 12 h 52

After blood collection, the right ventricle of each mouse was perfused with saline and the right lung was harvested and fixed buffered formalin (10%, pH 7.2) for 24 h for histology analysis. The left lung was homogenized in RIPA buffer (20 mM Tris/HCl, 138 mM NaCl, 10% glycerol and 1% Triton) containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Mannheim, Germany) and centrifuged. The supernatant was stored at −80 °C. The total protein in the samples (tissues and BAL) was determined with bicinchoninic acid (BCA) colorimetric assay (Thermo Scientific, Rockford, IL, USA). The lung proteins were analyzed for quantification MMP-12 (H-52; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and total β-actin (CP01; Calbiochem, San Diego, CA, USA) by Western blotting.

Total protein (30 µg/lane) from lung homogenate was subjected to 8 or 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then washed once with Tris/HCl, pH 7.4, containing 159 mM NaCl and 1% Tween 20, Tris buffered saline (TBS), and then blocked with 5% non-fat milk (Nestlé, São Paulo, Brazil). After, the membranes were washed with TBS and probed with MMP-12 (H-52; Santa Cruz Biotechnologies, Santa Cruz, CA, USA). β-actin (#5441; Sigma-Aldrich, St. Louis, MO, USA; 1:1000) was used as a loading control. After washing with TBS-T, following incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000), after washing with TBS, immunoreactive protein bands were revealed using an enhanced chemiluminescence assay (Amersham Pharmacia, Little Chalfont, UK). The results are expressed as arbitrary units.