The practical use of the optical fractionator. The optical fractionator design was applied on 7-9 coronal sections covering the whole dentate gyrus. The sections were double-stained for BrdU and ED-1, mounted on glass slides, and cover-slipped with Pertex® (Bie & Berntsen, Rødovre, Denmark) and thin cover glasses. CAST® software (Visiopharm A/S, Hørsholm, Denmark) was used for the computer assisted counting. A shows an example of one of the coronal sections used for the counting of BrdU+ cells. First, the SGZ of the DG was delineated with a 4× objective (B). A defined area sampling fraction (asf) within the delineated area was used for the counting procedure applying the principle of systematic unbiased random sampling. The asf was determined based on test counts where 150-200 BrdU+ cells in each hemisphere were counted. The CAST® software generated an unbiased counting frame as shown in C. BrdU+ cells within the frame or intersecting the green inclusion lines were counted using a 40× objective. All cells intersecting or touching the red exclusion lines were not counted. The scale bars represent 5 mm (A), 500 μm (B), and 100 μm (C), respectively. asf, area sampling fraction; BrdU, 5-bromo-2'-deoxy-uridine; DG, dentate gyrus; SGZ, subgranular zone.