Polyclonal rabbit anti rat chemerin

Thoracic aortic tissues isolated at different stages were fixed in 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 at room temperature and then blocked by 5% BSA. Slides were incubated with polyclonal rabbit anti-rat chemerin (1 : 1000, Abcam, UK). Subsequently, tissues were treated with goat anti-rabbit immunoglobulin G (1 : 500, Invitrogen Life Technologies, USA) and stained by 4′,6-diamidino-2-phenylindole (DAPI). The confocal microscope (Leica, Germany) was employed to capture fluorescence images. All parameters were measured by computer-assisted color image analysis.

Total proteins (20 μg) extracted from thoracic aortas were resolved on 10% SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes by electrophoresis. The membranes were blocked in 5% nonfat milk and TBS containing 0.3% Tween-20, subsequently incubated overnight with polyclonal rabbit anti-rat chemerin (1 : 1000, Abcam, UK), NF-κBp65 (1 : 1000, Cell Signaling Technology, USA), PCNA (1 : 500, Santa Cruz, USA), p-p38-MAPK (1 : 1000, Santa Cruz, USA), p-ERK 1/2 (1 : 1000, Santa Cruz, USA), p-JNK (1 : 500, Cell Signaling Technology, USA), and β-actin (1 : 1000, Santa Cruz, USA). The blots were incubated in 1 : 6000 dilution of goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies. 100 μl ECL solution was added in each sample to detect the protein signal with the Quant RT ECL cold CCD imaging system (General Electric, USA).