All brains were coded throughout staining and quantification procedures so that experimenters were blind to the treatment groups. Quantification of immunohistochemistry and staining was conducted on an Olympus BX-51 microscope with a motorized stage, and 12 MP camera (Olympus; Center Valley, PA). DCX-immunoreactivity (DCX+IR) was quantified at 100X in the dorsal dentate gyrus plus subgranular zone (an approximate 50μm ribbon between the granule cell layer and the hilus) as pixels/μm2 via image analysis system as described previously (Image Pro Plus, Bethesda, MD; Nixon and Crews, 2004). Profile counting methodology was used for all other histological measures and utilized a 40X lens for FJB (Olympus Plan Apo, numerical aperture 0.9) and a 60X oil immersion lens for all others (Olympus Plan Apo, numerical aperture 1.4). Profile counting methodology was chosen as these cell populations are nonhomogenously distributed and frequently total less than 100 profiles per region sampled, which suggests that stereology is not the best approach for quantification (Popken and Farel, 1997). Further, we have previously shown that profile counts and stereological estimates result in identical percent change and relative difference is the data of interest in these studies (Crews et al., 2004).
