To examine the various components of neurogenesis, immunohistochemistry with diaminobenzidine (DAB) detection was conducted on free-floating brain sections as described previously (Nixon and Crews, 2002; Nixon and Crews, 2004). Doublecortin (DCX, 1:400; Santa Cruz, Santa Cruz, CA), a well-accepted marker of immature neurons, was used to examine neurogenesis on every 12th section (Brown et al., 2003; Rao and Shetty, 2004). Cell proliferation and new cell survival were examined by manipulating the timing of BrdU injection versus sacrifice. BrdU (1μg/ml; Chemicon, Temecula, CA) immunohistochemistry was conducted on every 6th section of tissue and used a DNA denaturing step as described previously (Kuhn et al., 1996; Nixon and Crews, 2004). Ki-67 (1:200, Vector Labs, Burlingame, CA), an endogenous marker of proliferation that labels cells in all active phases of the cell cycle, was conducted on every 12th section of tissue (Scholzen and Gerdes, 2000). With the exception of the DNA denaturing step for BrdU, assays followed the same basic protocol as follows: cryoprotectant was rinsed off in tris buffered saline (TBS, Bio-Rad, Hercules, CA) and endogenous peroxidases were quenched by 0.3% H2O2
