BrdU (65 mg/kg, Sigma-Aldrich, Missouri, USA) was administered via intraperitoneal injection once daily on P56 and P57. One hour after the last injection, mice were anesthetized with 250 mg/kg tribromoethanol (Sigma-Aldrich, Missouri, USA) and then transcardially perfused with cold 1X PBS followed by 4% paraformaldehyde (PFA; Sigma-Aldrich, Missouri, USA). The brains were extracted and transferred to 4% PFA to postfix for 1 hour and then stored at 4°C in 20% sucrose for cryoprotection. The brains were cryosectioned (20-μm sections) on superfrost slides (Fisher Scientific, Pennsylvania, USA) and maintained at 4°C until staining.

Slides were thawed for 10 min at room temperature and rehydrated with 1X PBS followed by permeabilization with 0.03% Triton X-100 (Sigma-Aldrich, Missouri, USA). Slides were incubated in 1 N HCl at 65°C for 30 min and then in 10% normal donkey serum (NDS; Sigma-Aldrich, Missouri, USA) for 1 hour at room temperature. Next, slides were incubated with primary rat BrdU antibody (1:100, Abcam, Cambridge, UK) overnight followed by a donkey anti-rat Alexa Fluor 488 secondary antibody (1:400, Invitrogen, Oregon, USA) for 2 hours at 37°C and then coverslipped with Vectamount mounting media (Vector, California). Quantification was performed at ×20 magnification by counting one section per every 200 μm beginning at the crossing of the corpus callosum and ending at the appearance of the anterior commissure. Approximately four to six sections were counted per brain. BrdU+ cells were counted on a Zeiss microscope (Axiovert 200 M, Zeiss, Germany).

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