Coronal brain sections (40-μm thick) were prepared from injected mice and processed for immunostaining as described (Ge et al., 2006). The following primary antibodies were used: DCX (goat, 1:250, Santa Cruz), GFP (rabbit, 1:1000, Abcam), Prox-1 (rabbit, 1:1000, Abcam), Parvalbumin (mouse, 1:2000, Sigma), pAKT (rabbit, 1:50, Cell Signaling), pS6 (rabbit, 1:1000, Cell Signaling). Effective immunostaining of pAKT and pS6 required an antigen retrieval protocol. The sections were incubated for 30 min in 4’,6’-diaminodino-2-phenylindole (DAPI, 1:5000) before washing and mounting. Images were acquired on a META multiphoton confocal system (Zeiss LSM 510) using a multi-track configuration. For comparison of pAKT and pS6 levels, sections were processed in parallel and images were acquired using the identical settings. Fluorescence intensity was measured within the cytosol of individual GFP+DCX+ neuron and the value was normalized to those of GFP-DCX+ neurons in the same image for each neuron. All experiments were carried out in a blind fashion to experimental conditions. Statistic significance was determined by ANOVA.
