Diaminobenzidine dab staining

Eighteen to 24 hr after their last fluoxetine treatment or defeat exposure, animals were anesthetized with chloral hydrate and perfused intracardially with 200 ml of PBS (11.9 mM phosphates, 137 mM NaCl, 2.7 mM KCl; pH 7.4), followed by 400 ml of 4% paraformaldehyde in PBS. Brains were removed and stored overnight in 4% paraformaldehyde at 4°C, then transferred to a 30% sucrose in PBS solution and stored at 4°C until isotonic. Coronal sections (35 μm) were cut on a freezing microtome (Leica, Bannock-burn, IL) and then processed for immunohistochemistry as described (Perrotti et al., 2008 (link)) using a polyclonal FosB antibody (SC-48, Santa Cruz Biotechnology, Dallas, TX) which recognizes all three major FosB gene products. FosB positive cells were visualized using diaminobenzidine (DAB) staining (Vector Laboratories, Burlingame, CA) and counted by a double-blind experimenter. The number of FosB immunopositive cells was counted in the entire brain area in a given 35 μm slice and divided by the area to give cells/mm². FosB was quantified in several sections through the brain of each mouse, and mean values were then calculated for each mouse. Each mouse was considered an individual observation for statistical analysis. Brain regions were defined by (Paxinos and Franklin, 2004 ).