Bicinchoninic acid bca colorimetric assay

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.

Mice were euthanized and the brains were rapidly extracted from the skulls. Half-brains were homogenized in SDS-containing Lysis Buffer (2% SDS, 62.5 mM HEPES/NaOH, pH 8.0; preheated to 90°C), with the aid of 1.0 mm zirconia beads and the Mini-BeadBeater-8 (Biospec Products Inc., Oklahoma, USA). Following three one-minute cycles of bead beating, brain lysates were further incubated at 90°C to deactivate residual enzymatic activities in the extracts. The mouse blood was collected during transcardiac perfusion and solubilized in SDS-containing Lysis Buffer assisted by sonication with a Sonic Dismembrator device (Model 500, Thermo Fisher Scientific). For mouse serum preparation, blood was collected saphenously or transcardially and clotted in microvettes (CB300 Z, clotting/activator serum) (catalog number 16.440.100, Sarstedt, Nürnbrecht, Germany) at 4°C, followed by centrifugation (10,000 g, 5 min). Protein levels of the brain or blood samples were adjusted by bicinchoninic acid (BCA) colorimetric assay (Thermo Fisher Scientific). Identical volumes of serum samples were used for further analyses.