Mouse angiogenesis antibody array kit

Measurement of mouse adipokines and growth factors from fat tissue filtrates was performed using the Mouse Angiogenesis Antibody Array kit (R&D Systems Cat# ARY015). By comparing the signal intensities, relative expression levels of cytokines were determined and quantified by densitometry using ‘ImageJ’ software (NIH).

Skeletal muscles were harvested from non-ischemic, sham or ischemic hindlimbs of WT and Fgf2−/− mice. Sham or ischemic thigh and calf muscles were dissected free of the femur and tibia/fibula, respectively and snap-frozen in liquid nitrogen at 7 days of ischemia. 100–120mg of skeletal tissues was pulverized and homogenized on ice in a glass dounce homogenizer in an extraction buffer containing 20mM Tris, 2mM EDTA, 2M NaCl, 1% NP40, EDTA-free protease inhibitor cocktail (Roche, 1 tablet/10mL), and 1mM PMSF. The homogenate was centrifuged at 15,000g for 15 minutes and the supernatant was collected. After protein concentration was determined using a Bio-Rad DC protein assay, 600µg of total protein in 1.5mL of array buffer from the proteome profiler mouse angiogenesis antibody array kit (R&D Systems) was mixed with 15µL of a detection antibody cocktail and incubated at 25°C for one hour. Each sample/antibody mixture was then added to a nitrocellulose membrane containing 52 different capture antibodies (listed in Table 2) dotted in duplicate and incubated at 4°C overnight. After washing, 2mL of buffered streptavidin-HRP was added to each membrane and allowed to incubate at 25°C for 30 minutes. Array membranes were developed using chemiluminescence with the provided Chem Reagent mix. The densitometry of the antibody/protein dots were quantified using a Fluorchem 8800 gel imager.