Immunohistochemistry
At 2 h after the last challenge, when expression levels of c-Fos protein are at their peak (Zangenehpour and Chaudhuri, 2002), mice were deeply anesthetized with an overdose of sodium pentobarbital (200 mg/kg) and transcardially perfused with saline followed by 4% paraformaldehyde (in 0.1 mol/l phosphate buffer, pH 7.4), as described previously (Muigg et al, 2009). One-in-eight of the free-floating coronal sections (50 μm) throughout the entire murine HPC were processed for BrdU, doublecortin (DCX), and c-Fos immunohistochemistry, respectively, following established protocols (Couillard-Despres et al, 2009; Muigg et al, 2009). Sections were incubated with one of the following primary antibodies: rat anti-BrdU (1 : 350; Serotec, Dusseldorf, Germany), goat anti-DCX C18 (1 : 250; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti c-Fos (1 : 10 000; Santa Cruz Biotechnology). Subsequently, they were incubated with the corresponding biotinylated goat anti-rat, rabbit anti-goat, or goat anti-rabbit secondary antibody (all 1 : 200; Vector laboratories, Burlingame, CA). The formed antigen-antibody-complexes were visualized by the avidin-biotin-horseradish peroxidase procedure (Vectastain Elite ABC kit; Vector Laboratories) using 3,3′-diaminobenzidine as the chromogen.
Immunoreactive cells were quantified in the regions of interest using a light-optical microscope (Olympus, Vienna, Austria) and a computer-assisted image analysis system (cellSens Dimension; Olympus). A cell was considered as labeled (positive) for c-Fos, DCX, or BrdU when the brown-black DAB staining was unambiguously darker than the background, and this included all cells from low to high intensities of staining.