Immunohistochemical Analysis of c-Fos Expression

The c-Fos immunohistochemistry was performed as previously described^{53 (link)}. Briefly, anesthetized male mice were intracardially perfused with cold PBS followed by a cold 4% paraformaldehyde (PFA) in PBS. The brains were removed and post-fixed in a 4% PFA solution overnight at 4 °C. Brain regions were cut into 2–4 large blocks. The blocks were sliced on a microtome into 20-μm-thick sections. The sections were pre-incubated in blocking solution (3% bovine serum albumin and 0.3% Triton X-100 in PBS) for 1 h, then incubated with an anti-c-Fos antibody (sc-52, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the blocking solution for 12 h at 4 °C. After three washes with washing buffer, the sections were incubated with goat anti-rabbit IgG antibody coupled with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) in the blocking solution for 1 h at room temperature. The images were obtained by using an Olympus IX71 inverted microscope equipped with a cooled CCD camera (Cool SNAP HQ2; Roper Scientific, Tucson, AZ, USA). The number of c-Fos immuno-positive nuclei in each brain section were recorded and analysed using Metamorph software (Molecular Devices, Downingtown, PA, USA).