c-Fos immunohistochemistry

SD rats (8 weeks old, baseline BW 291 ± 14 g) fed HFD for 2 weeks were randomized based on BW, and were administered drugs (N = 4 per group, total N = 16). Rats were dissected 3 h after the dosing to isolate the brain stem under sodium pentobarbital (50 mg/kg, i.p., Kyoritsu, Tokyo, Japan) anesthesia. The obtained brain samples were fixed with 4% paraformaldehyde for 24 h, followed by substitution with 30% sucrose in phosphate-buffered saline (PBS) for 2 days. Immunostaining was performed by a free-floating method. Forty μm cryosections including NTS were incubated with anti-cFos rabbit polyclonal antibody (SC-52; Santa Cruz Biotechnology) diluted 1:4000 in PBS at pH 7.1 containing 1% normal horse serum and 0.4% Triton X-100 overnight. Immunoreactivity in the sections was visualized using diaminobenzidine after the treatment with an ABC Elite kit (PK-6101, Vector Laboratories, Inc., Burlingame, CA). After processing, the sections were mounted on a non-coated slide, dehydrated, and then cover-slipped. Photographs were captured with a 4 x objective lens using Nikon ECLIPSE 800 microscope equipped with Nikon ACT-1 version 2.12. c-Fos positive cells were counted using a computerized image analysis system (Image-Pro Plus version 4.5). c-Fos counts were performed on each of three sections from each animal. The mean of these three determinations was used for subsequent statistical analysis. In the same protocol as described above, Ffar1^-/- and wild-type mice (46 weeks old, baseline BW 50 ± 5 g for wild and 55 ± 2 g for Ffar1-/-) fed HFD for 38 weeks were also used to confirm the GPR40 dependency (N = 5–6 per group, total N = 11 in each genotype).