Rabbit antibody to artemin

Total protein was extracted using radioimmune precipitation buffer protein lysis buffer according to protocols (Beyotime, Shanghai, China). The protein content of the samples was measured by means of the BCA protein assay kit (Beyotime, Shanghai). The protein samples were denatured and separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The nitrocellulose membranes were blocked for 1 h at room temperature in 5 % skimmed dried milk. Then the membranes were incubated with primary antibodies including: rabbit antibody to GFRα3 (1:1000, Abcam Systems, USA), rabbit antibody to artemin (1:200, Abbiotec, San Diego) and mouse antibody to β-actin (1:1000, Santa Cruz Biotechnology, USA) in TBST containing 3 % fat-free dry milk for 1 h at room temperature and overnight at 4 °C. After washing 3 times with TBST, the membranes were incubated with the secondary donkey anti-rabbit or anti-mouse IgG antibodies (1:5000, Santa Cruz, USA) at room temperature for 1 h. Finally, the immunoblots were detected using an ECL kit (Santa Cruz, USA) and visualized after exposure to X-ray films. The relative optical density ratio was calculated with the Image J software by comparison to GAPDH or β-actin.

Tissues were fixed in 4 % paraformaldehyde at 4 °C for 24 h, followed by incubated in 30 % sucrose for 24 h, embedded in optimum cutting temperature compound for 24 h, quickly frozen with liquid nitrogen, and cut into 6 μM sections using a cryostat (Leica CM 1850, Nussloch, Germany). For immunofluorescence, samples were washed in 0.01 M PBS and blocked in PBS containing 0.3 % Triton X-100 (Sigma, USA) and 10 % donkey serum (NQBB, USA) for 1 h at room temperature and incubated in primary antibodies overnight at 4 °C. The following antibodies were used: goat antibody to GFRα3 (1:50, R&D Systems, USA), rabbit antibody to artemin (1:50, Abbiotec, San Diego) and mouse antibody to α-smooth muscle actin (α-SMA) (1:50, Dako Cytomation, Germany), After being washed fully in PBS, samples were incubated with secondary antibodies (1:1000, Invitrogen, USA) and 49,6-diamidino-2-phenylindole (DAPI) (1:1000, Molecular Probes, USA) in PBS at room temperature for 1 h. Then rinsed in PBS for 30 min. Immunofluorescent samples were visualized with an inverted DMI 400CS confocal microscope (Leica, Germany).