Immunohistochemistry of free-floating brain sections was performed as described previously (Nixon & Crews, 2004), using monoclonal anti-mouse BrdU antibody (1 :2000, MAB3424, Chemicon, USA), monoclonal mouse anti-Ki67 [1 :200; Novocastra Laboratories, UK (no. NCL-Ki67-MM1)], polyclonal goat anti-doublecortin antibody (anti-DCX, 1: 400, SC8066, Santa Cruz Biotechnology, USA) and polyclonal rabbit anti-Sry-related high-mobility group box 2 antibody (anti-SOX2, 1: 200, AB5770, Chemicon). To control for day-to-day variability, controls and alcohol-exposed animals for their respective time-point were always processed in parallel.
