Bead ruptor 4 homogenizer

Mice were sacrificed by CO₂ inhalation, and the vasculature was perfused with phosphate-buffered saline, via the left ventricle, to remove all blood. The aortas from E19, P15, and P90 were then carefully excised (free of fat) from the root down to the diaphragm. Vessels were snap frozen in liquid nitrogen and stored at −80 °C. For Western blot analysis, the frozen aortas were then homogenized using the Bead Ruptor 4 Homogenizer (Omni International) in 200 µL of RIPA buffer (Milliporesigma, R0278, Burlington, MA, USA) containing complete EDTA-free Protease Inhibitor Cocktail (MilliporeSigma, 11873580001, Burlington, MA, USA), and protein was quantified with Pierce Rapid Gold BCA Protein Assay kit (Thermo Scientific, A53226). A total of 30 µg of total protein was loaded on TGX stain-free protein gels (Bio-Rad, 17000927, Hercules, CA, USA) for blotting.

The aorta, from the root to diaphragm, was dissected out and snap frozen in liquid nitrogen. Lox enzyme activity was measured in tissues as described by Trackman and Bais [19 (link)] by measuring the production of hydrogen peroxidase through oxidation of Amplex Red (ThermoFisher Scientific, A12222, Waltham, MA, USA), which results in the generation of highly fluorescent resorufin for detection. Briefly, the frozen aortas were homogenized using the Bead Ruptor 4 Homogenizer (Omni International) in 250 µL of buffer containing 6M urea and 50 mM borate (pH 8.2). One hundred microliters of samples were then added to the reaction buffer with final concentration of 1.2M urea, 50 mM borate (pH 8.2), 1 unit/mL of horseradish peroxidase, 12.5 µM Amplex Red, and 12.5 mM 1,5-diaminopentane. Each sample was tested in duplicate. Parallel assays were prepared with 625 mM β-aminopropionitrile (BAPN) to inhibit the Lox activity. All reactions were incubated at 37 °C and measured for fluorescence at excitation wavelength of 563 nm and emission wavelength at 587 nm continuously for 60 min in CLARIOstar microplate reader (BMG Labtech). Reported activity is the average slope of the line generated from plotting the resorufin fluorescence by time done in n = 8.