4-day old adult male brains were dissected in a phosphate-buffered saline solution (PBS) and fixed for 45 min at room temperature with 4% formaldehyde (Ted Pella, Inc, Redding, CA). Tissue was left overnight at 4°C with 1:200 goat rabbit anti-GFP (Invitrogen Molecular Probes, Eugene, OR) and 1:50 goat anti-mouse nc82 (Jackson Laboratories, Sacramento, CA), washed four times and left overnight in 1:500 Alexa-Fluor 594 goat anti-mouse (Invitrogen Molecular Probes, Eugene, OR) and 1:200 Cy2 goat anti-rabbit (Jackson Laboratories, Sacramento, CA). Brains were mounted in Fluoromount-G (Southern Biotech, Birmingham, AL).
