Alexa fluor 568 donkey anti sheep

Adult brains were dissected out and fixed with 4% paraformaldehyde for 20 min. After that, samples were rinsed with PBT (PBS 1× +0.3% Triton X-100) and then blocked with BSA 1% in PBT + sodium azide 0.03% for 20 min. Tissue was incubated with sheep polyclonal anti-TPH/Trh antibody (AB1541, Millipore, Burlington, MA, USA) (1:50) for three days at 4 °C to identify serotonergic neurons or with rabbit polyclonal anti-TH antibody (AB152, Millipore, Burlington, MA, USA) (1:300) for one day at 4 °C to identify dopaminergic neurons. The use of the anti-TPH antibody in immunohistochemistry was previously validated by Bao and colleagues [24 (link)]. Then, samples were rinsed with PBT and incubated for 2 h at room temperature with secondary Alexa Fluor 568 donkey anti-sheep (A-21099, Thermo Fisher Scientific, Waltham, MA, USA) (1:500) or Alexa Fluor 568 goat anti-rabbit (A-11011, Thermo Fisher Scientific, Waltham, MA, USA) (1:500) to visualize serotonergic or dopaminergic neurons, respectively. Finally, brains were rinsed with PBT and mounted using SlowFade™ Gold Antifade Mountant with DAPI (S36938, Thermo Fisher Scientific, Waltham, MA, USA).