Mab377

NeuN immunohistochemistry (Fig 2) was performed using a mouse anti-NeuN antibody (1:2000; Millipore, ref. MAB377) as the primary antibody, and a biotinylated anti-mouse horse antibody (1:500; Vector Laboratories, ref. BA-2001) as the secondary antibody. Briefly, sections were first rinsed three times for 10 min in PBS before being soaked for 1h in 5% normal donkey serum in PBS containing 0.5% Triton X-100. The sections were transferred to the primary anti-NeuN antibody solution for 18h at room temperature, then rinsed three times and soaked in a buffer solution containing the biotinylated secondary antibody for 1h. After three more rinses, the staining was finally amplified using the avidin-biotin peroxidase method (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) and revealed using diaminobenzidine (SK-4100 kit, Vector Laboratories). The second half of the brain sections from the region including the ventral midline thalamus were stained with Cresyl violet (Fig 2). These brain sections were directly collected on slides, contrary to their NeuN counterparts. The successive baths for Cresyl violet staining were as follows: distillated water (1 min); 0.1% Cresyl Violet for (12 min); 70% ethanol (2 min); 95% ethanol (2 min) and 100% ethanol (2 min). Finally, slides were defatted in 100% Xylene (2 times 10 min) before being mounted on DPX medium.