Immunohistochemistry of Arc

Immunohistochemical assay was performed as described by our previous study (Li et al., 2009). Briefly, the brain slices from different groups were cut at a thickness of 30 μm and washed with 0.1 mol/L phosphate buffer. The brain slices were blocked with 10% normal goat serum for 2 h at room temperature and then incubated overnight with primary antibody (mouse anti-Arc antibody, 1:500 dilution in 10% normal goat serum) at 4°C. Next, the brain slices were incubated with secondary antibody (biotinylated goat anti-mouse IgG, 1:200 dilution in 10% normal goat serum) for 2 h at room temperature. Arc-positive sites were visualized using a streptavidin-ABC kit and a DAB kit using 0.1% DAB as the chromogen. In control slices in which the primary antibodies were omitted or replaced by nonimmune rabbit or goat serum, no stained cells were seen. The brain slices were subsequently dehydrated in alcohol and xylene, coverslipped, and imaged on an Olympus IX51 microscope.