Polyclonal rabbit

Brain-tissue samples were collected using 2-deoxy-d-glucose, ethylene glycol tetra-acetic acid (EGTA), and reduced glutathione. Western blot and immunofluorescence analyses involved primary antibodies; primary monoclonal rat anti-tyrosine hydroxylase (T1299, Sigma-Aldrich, St. Louis, MI, USA), monoclonal rabbit anti-NPY5R (AB133757, Abcam, Cambridge, UK), polyclonal rabbit AMPKα1/2 (H-300) (sc-25792, Santa Cruz, CA, USA), polyclonal rabbit anti-phospho AMPKα (THR172, Millipore, Burlington, MA, USA), monoclonal mouse anti-β-actin (A5441, Sigma-Aldrich, St. Louis, MI, USA), and polyclonal rabbit anti-tyrosine hydroxylase phosphoSer40 (AB5935, Millipore, Burlington, MA, USA). Primary antibodies in Western blot analysis were detected using polyclonal goat anti-mouse IgG HRP (HAF007, R&D Systems, Minneapolis, MN, USA) and polyclonal goat anti-rabbit IgG (H + L) HRP (65-6120, Invitrogen, Waltham, MA, USA), whereas Alexa Fluor 488 conjugated with goat anti-rabbit IgG (H + L) secondary antibody (A11008, Thermo Fisher Scientific, Waltham, MA, USA) and CY3 conjugated with goat anti-mouse IgG (H + L) secondary antibody (A10521, Thermo Fisher Scientific, Waltham, MA, USA) were used in immunofluorescence.

Immunohistochemical staining for SRF was performed using a microwave-induced antigen retrieval method as previously described [26 (link)]. De-waxed sections were immersed in a citric acid buffer and placed in a 700 W microwave oven at full power for 15 min. Using a standard avidin-biotin complex method (Vector Laboratories, Inc.), the sections were incubated with polyclonal rabbit (Santa Cruz Biotechnology, Inc. – 1:800 dilution) at 4 °C overnight. The colour reaction product was obtained with DAB and counterstained with Haematoxylin. Tonsil sections were used as positive controls. Prior to this study, the SRF antibody was subjected to western blot analysis using LNCaP and PC-3 prostate cancer cell lines which confirmed specificity for SRF [6 (link)].