Immunohistochemical Analysis of c-Fos Expression

The sagittal sections containing the major downstream target regions of the rat prostriata were selected for c-fos IHC. These regions include the PrSd-PoS, LD, LP-Pul, PTN, and VLG and are directly innervated by the prostriata (Chen et al., 2021 (link)). In addition, we also evaluated c-fos expression in zona incerta (ZI) and substantia nigra (SN), which are not the targets of the prostriata. Selected sections were rinsed three times with 0.1 M PB and immersed in 0.3% hydrogen peroxide for 10 mins. Then the sections were rinsed three times again and blocked in 5% BSA for 60 mins. At the end, the sections were incubated with primary antibody against c-fos (200 μg/ml, 1:200, Boster Biological Technology, Wuhan, China) diluted with the 0.1 M PB overnight at 4°C. On the next day, the sections were incubated in the secondary antibodies (biotinylated goat anti-mouse/rabbit IgG, Boster) for 60 mins after thorough rinse with 0.1 M PB. The sections were then rinsed again and immersed in the Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 60 mins. After rinse with 0.1 M PB, the sections were incubated in 3, 3-diaminobenzidine (DAB) solution for 3 mins in a dark environment. Finally, the sections were mounted on the slides, dehydrated and coverslipped.