Rabbit anti vegf a

The glomeruli were isolated using a sieve, lysed in buffer (20 mM Tris, 140 mM NaCl, 2 mM EDTA, 10% glycerol, and 0.5% Triton X-100) containing protease inhibitor cocktail for 30 min, and centrifuged at 13,000× g at 4 °C for 20 min. Equal amounts of total protein were denatured (98 °C, 5 min) and then electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins were transferred to PVDF membranes (2 h at 200 mA). The membranes were blocked (5% BSA, 0.05% Tween-20 in TBS) for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with a primary antibody (rabbit anti-VEGF-A, 1:500; Merck KGaA, Darmstadt, Germany, Cat. No. AB1876-I; rabbit anti-β-actin, 1:1000, Sigma-Aldrich, Saint Louis, MO, USA, Cat. No. A2066). Membranes were washed (0.01% Tween-20 in TBS) and exposed to horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG; BD Pharmingen, Franklin Lakes, NJ, USA, Cat. No. 554021) for 1 h at room temperature. Super Signal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect reaction products. The GelDoc-It imaging system (UVP) was used to analyze and archive the membranes. The background signal was subtracted using the rolling disk method.

The glomeruli were isolated using the sieving technique and then lysed in buffer (20 mM Tris, 140 mM NaCl, 2 mM EDTA, 10% glycerol and 0.5% Triton X-100) containing Protease Inhibitor Cocktail for 30 min and centrifuged at 13,000× g for 20 min at 4 °C. Identical amounts of total protein were denatured (98 °C, 5 min) and then subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The proteins were transferred to membranes (2 h at 200 mA) and the membranes were blocked (5% BSA, 0.05% Tween-20 in TBS) for 1 h at room temperature. Next, the membranes were incubated at 4 °C overnight with primary antibody (rabbit anti-VEGF-A, 1:500; Merck KGaA, Cat. #AB1876-I; rabbit anti-β-actin, 1:1000, Sigma-Aldrich, A2066). After washing (0.01% Tween-20 in TBS), secondary antibodies conjugated to horseradish peroxidase (anti-rabbit IgG; BD Pharmingen, 554021) were added to the membranes for 1 h at room temperature. The reaction products were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were analysed and archived in a GelDoc-It Imaging System (UVP). The rolling disc method was used to subtract the background signal.