Immunohistochemical Detection of Serotonin Pathway

As previously described method (Park et al., 2019 (link)), immunohistochemistry for TPH and 5-HT in the dorsal raphe was performed. The sections were incubated in PBS for 10 min, and then washed 3 times in the same buffer. The sections were then incubated in 1% H₂O₂ for 20 min. The sections were selected from each brain and incubated overnight with mouse anti-TPH antibody (Abcam, Cambridge, UK) and rabbit anti-5-HT antibody (Abcam) at a dilution of 1:200 for TPH and 5-HT expression. The sections were incubated for 1 hr with biotinylated anti-mouse secondary antibody or with biotinylated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA). They were subsequently incubated with avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hr. Immunoreactivity was visualized by incubating the sections with a solution consisting of 0.05% 3,3′-diaminobenzidine and 0.01% H₂O₂ in 50 mM Tris-buffer (pH, 7.6) for approximately 3 min. The sections were finally mounted on gelatin-coated glass slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount (Thermo Fisher Scientific Inc., Waltham, MA, USA).