Biotinylated anti rabbit secondary antibody

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).

Immunohistochemistry for 5-HT and TPH was performed, according to the previously described method (Shin et al., 2017 (link)). An average of eight sections was selected in each brain region spanning from Bregma −7.30 to −8.00 mm. The sections were incubated in PBS for 10 min and they were next washed three times with PBS. The sections were then incubated in 1% H₂O₂ for 30 min, and then they were incubated overnight with rabbit anti-5-HT antibody (Oncogene Research Product, Cambridge, UK) at a dilution of 1:500 or with mouse anti-TPH antibody (Oncogene Research Product) at a dilution of 1:500. The sections were incubated for 1 hr with biotinylated anti-rabbit secondary antibody or with anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA), and they were subsequently incubated with avidin–biotin–peroxidase complex (Vector Laboratories) for 1 hr at room temperature. Immunoreactivity was visualized by incubating the sections in 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).

For immunolabeling of c-Fos in the PVN and vlPAG, c-Fos immunohistochemistry was performed as the previously described method (Han et al., 2017 (link); Ko et al., 2016 (link)). Free-floating tissue sections were incubated overnight with rabbit anti-c-Fos antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the section were then incubated for 1 hr with biotinylated anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). The sections were subsequently incubated with avidin-biotin-peroxidase complex (1:100; Vector Laboratories) for 1 hr at room temperature. Immunoreactivity was visualized by incubating the sections in 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. After washing three times with PBS, the section was mounted onto gelatin-coated slides. At room temperature, the slides were air-dried overnight, and coverslips were mounted using Permount (Fisher Scientific).

Immunohistochemistry for 5-HT and TPH was performed, according to the previously described method (Shin et al., 2017b (link)). An average of eight sections was selected in each brain region spanning from Bregma −7.30 to −8.00 mm. The sections were incubated in PBS for 10 min and they were next washed three times with PBS. The sections were then incubated in 1% H₂O₂ for 30 min, and then they were incubated overnight with rabbit anti-5-HT antibody (Oncogene Research Product, Cambridge, UK) at a dilution of 1:500 or with mouse anti-TPH antibody (Oncogene Research Product) at a dilution of 1:500. The sections were incubated for 1 hr with biotinylated anti-rabbit secondary antibody or with anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA, USA), and they were subsequently incubated with avidin–biotin–peroxidase complex (Vector Laboratories) for 1 hr at room temperature. Immunoreactivity was visualized by incubating the sections in 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).