Biotinylated anti rabbit 5 ht secondary antibody

Immunohistochemistry was conducted to evaluate the 5-HT and TPH expression in the dorsal raphe, according to the previously described method (Ji et al., 2017 (link); Moon et al., 2018 (link)). Free-floating tissue sections were incubated overnight with mouse anti-TPH or rabbit anti-5-HT antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were treated with biotinylated, anti-mouse TPH secondary antibody or with biotinylated anti-rabbit 5-HT secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) during 1 hr. Next, avidin-biotin-peroxidase complex (Vector Laboratories) was treated to the section during 1 hour at 25°C. The sections were treated with a reaction mixture consisting of 0.03% 3,3′-diaminobenzidine and 0.03% hydrogen peroxide during 5 min. After the slides were dried under the room conditions, Permount (Fisher Scientific, Fair Lawn, NJ, USA) was used for the coverslips mounting.

As the method described previously [20 (link),23 (link)], we performed immunohistochemistry to detect the expression of 5-HT, TPH, and TH. The tissue sections were treated with mouse anti-TH, mouse anti-TPH, and rabbit anti-5-HT antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) during overnight. The sections were treated with biotinylated anti-mouse TH, anti-mouse TPH secondary antibody and with biotinylated antirabbit 5-HT secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) during 1 hour. Next, avidin-biotin-peroxidase complex (Vector Laboratories) was treated to the section during 1 hour at 25℃. The sections were treated with a reaction mixture consisting of 0.03% 3,3’-diaminobenzidine and 0.03% hydrogen peroxide during 5 minutes. After the slides were dried under the room conditions, Permount (Fisher Scientific, Fair Lawn, NJ, USA) was used for the coverslips mounting.