Hypothalamic Neuronal Activation Mapping

To determine which nuclei in the hypothalamus were responsive to test-pellet consumption, we used c-Fos as a marker of neuronal excitation, as previously performed in a laboratory [26 (link)]. Brains were flash-frozen in methylbutane on dry ice and then sectioned at 30 microns using a freezing–sliding microtome. Sections were washed in potassium-phosphate-buffered saline (KPBS), blocked with 2% donkey serum in KPBS with 0.4% Triton x-100 in KPBS, and incubated with the primary antibody for c-Fos (1:2500, #ab190289, Abcam, Cambridge, MA, USA) overnight for two nights at 4 °C. The sections were then incubated with the anti-rabbit biotinylated secondary antibody (1:500, #BA1100, Vector Laboratories, Burlingame, CA, USA) for 60 min at room temperature, followed by A/B solution (1:600, #PK-6100, Vector Laboratories) for 60 min at room temperature. The final step was an incubation of 0.2 mg/mL DAB substrate (D5905, Sigma, Burbank, CA, USA) and hydrogen peroxide for 15 min at room temperature. Sections were then mounted on gelatin-coated slides.