Antibody Characterization:
Goat polyclonal antiserum against cholera toxin b subunit (CTb) (List Biological Laboratories, Campbell, CA). Kishi and colleagues (Kishi et al., 2000) demonstrated that this antibody stains CTb injection sites and retrogradely labeled neurons in the rat brain. As well, in present study this antibody clearly stained CTb injection sites and retrogradely labeled neurons in the mouse brain, but it did not stain brain sections from animals without CTb injections (data not shown), indicating that this antibody has no cross-reactivity to any of endogenous antigens in the mouse brain. The antibody is registered in the JCN Antibody Database and has been frequently and successfully used in many other studies (Al-Khater et al., 2008; Howorth et al., 2009; Wittmann et al., 2009)
Stereotaxic surgery for retrograde neuronal tracing:
Surgical procedure was followed as previously described (Chamberlin et al., 1998; Gautron et al., 2010a). Briefly, MC4R-GFP mice were anesthetized with ketamine HCl/xylazine HCl (80:12 mg/kg, i.p.) and restrained in a Kopf (Tujunga, CA) stereotaxic apparatus. A small hole was drilled into the skull under aseptic conditions. A glass micropipette connected to an air pressure injector system or iontophoresis machine was positioned via the stereotaxic manipulator. Approximately 50100 nL of retrograde tracer, fluorogold (Fluorochrome, CO, USA) or cholera toxin subunit B (CTb) (List Biological Laboratories), was slowly administered into the ventral tegmental area (VTA) (3.2 mm from bregma, 1.1 mm lateral with 10 degree injection arm, …
• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt
• Product #703 – Anti-Cholera Toxin B Subunit (Goat)