Rabbit anti ccl2 antibody

Proteins were extracted from Retina/RPE/Choroid tissues. The protein concentration was determined using the BCA protein assay kit. Equal amounts of proteins were resolved by SDS-polyacrylamide gel electrophoresis using 12% denaturing gels and then transferred onto polyvinylidene fluoride membranes (Millipore, USA). The blotted membranes were incubated with a rabbit anti-CCL2 antibody (1:2000; Abcam, UK) or rabbit anti-β-actin antibody (1:200; Boshide, China) at 4 °C overnight. The membranes were washed with Tris-buffered saline/Tween (TBS/T) three times and then incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000; Zhong Shan Jin Qiao, China) at room temperature for 2 hours. The membranes were washed again with TBS/T three times. Signals were developed by chemiluminescence using an ECL kit (GE, USA). The blots were visualized by the BioSpectrum Imaging System (UVP, USA) and analyzed by Gene Tool software (Syngene, UK).

Dot blot apparatus was assembled with nitrocellulose membrane and filter paper, following instructions of the manufacturer (Bio-Rad). SMC conditioned medium supernatant (100 μl) was applied to each Dot blot well, and the nitrocellulose membrane was allowed to dry for 1 h. The membrane was then blocked in 5% dry milk in tris-buffered saline (TBS) containing 0.1% Tween-20, and incubated in rabbit anti-CCL2 antibody (1:1000, Abcam) overnight at 4 °C. The next day, after washing in TBS with 0.1% Tween-20, the membrane was incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:2000; DAKO) for 1 h. Detection was performed with the Western Blotting Substrate Plus (Pierce) and GBOX imaging system (Syngene).