Immunohistochemistry, BrdU labeling analysis, and in situ hybridization

Immunohistochemical staining was performed as described in our previous studies^37,38. The following primary antibodies were used: mouse anti-BrdU (1:300; Calbiochem) or rat anti-BrdU (1:1000; Accurate Chemical & Scientific Corporation), goat anti-Calretinin (CR) (1:1000; Chemicon), goat anti-doublecortin (DCX) (1:400; Santa Cruz), goat anti-NeuroD (1:400; Santa Cruz), rabbit anti-Tph2 (1:20,000) (Gutknecht et al., 2008), rabbit anti-GFAP (1:500; Dako), rabbit anti-5-HTT (1:1000; gift from Dr. R.D. Blakely), rabbit anti-Ki67 (1:500; Abcam), mouse anti-MCM2 (1:1000; BD Pharmingen), goat anti-Sox2 (1:400; Santa Cruz), mouse anti-NeuN (1:1000; Chemicon) and rabbit anti-tyrosine hydroxylase (TH) (1:4000; Sigma).

For BrdU pulse labeling experiment to analyze cell proliferation, mice received 4 injections of BrdU at 50 mg/kg body weight at a 2-hour interval, and were sacrificed 2 hours after the last injection. For analysis of new-born cell survival, BrdU was injected before or after TM administration. In BrdU injection before depleting 5-HTergic neurons, BrdU was injected once daily for 3 consecutive days, followed by 4 times of TM administration; mice were sacrificed 30 days after the last TM administration. In BrdU injection after depleting 5-HTergic neurons, BrdU was injected 30 days after the last TM treatment for 3 consecutive days in the same way, and mice were allowed to survive for further 30 days after the BrdU injection. Brain sections were immersed in 0.01 M citrate buffer at 95 °C for 5 min, in 2 N HCl at 37 °C for 20 min and in 0.1 M sodium borate for 10 min, and then washed in PBS. Treated sections were immunostained with anti-BrdU antibody as described above.

AADC in situ hybridization was performed as described previously (Song et al., 2011). Images were captured with an epifluorescence (Eclipse 80i, Nikon) or confocal (TCS SP5, Leica) microscope.