Thirty micrograms of proteins obtained from all samples were processed as previously reported [35 (link)].
Membranes were incubated with primary antibodies rabbit anti-VEGF (1:750, rabbit; Sigma-Aldrich, Milan, Italy), RUNX2 (1:750, rabbit; Sigma-Aldrich), and anti-beta-actin (1:750, mouse; Santa Cruz Biotechnology, Santa Cruz, CA). After five washes in PBS 0.1% Tween-20, the tested samples were incubated for 1 h at room temperature with peroxydase-conjugated secondary antibody anti-rabbit and anti-mouse diluted 1:2000 in 1X PBS, 3% milk, 0.1% Tween [36 (link)]. Protein expression was analyzed using the enhanced chemiluminescence detection system (ECL) (Amersham Pharmacia Biotech, PA, USA) with photo documenter Alliance 2.7 (Uvitec, Cambridge, UK). Signals were taken by ECL enhancing and assessed utilizing an UVIband-1D gel analysis (Uvitec).
Membranes were incubated with primary antibodies rabbit anti-VEGF (1:750, rabbit; Sigma-Aldrich, Milan, Italy), RUNX2 (1:750, rabbit; Sigma-Aldrich), and anti-beta-actin (1:750, mouse; Santa Cruz Biotechnology, Santa Cruz, CA). After five washes in PBS 0.1% Tween-20, the tested samples were incubated for 1 h at room temperature with peroxydase-conjugated secondary antibody anti-rabbit and anti-mouse diluted 1:2000 in 1X PBS, 3% milk, 0.1% Tween [36 (link)]. Protein expression was analyzed using the enhanced chemiluminescence detection system (ECL) (Amersham Pharmacia Biotech, PA, USA) with photo documenter Alliance 2.7 (Uvitec, Cambridge, UK). Signals were taken by ECL enhancing and assessed utilizing an UVIband-1D gel analysis (Uvitec).
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