Fig. 2. Characterization of BrdU-labeled cells in the granule cell layer of the adult hippocampus. A, Typical BrdU-positive cells in the dentate gyrus of an adult rat hippocampus, as revealed with DAB visualized with Nomarski optics. Immunopositive nuclei are easily distinguishable and clearly separated, making them well suited for stereology counts. B, A higher magnification of the area marked in A to show BrdU+ nuclei in greater detail. Orientation inA is dorsal (D), ventral (V), medial (M), and lateral (L). C, A BrdU+cell (red nucleus) within the granule cell layer also shows IGF-I receptor labeling (green signal surrounding the nucleus). D, Representative BrdU+ nuclei in the hippocampus of an animal after 15 d of treadmill exercise and simultaneous infusion of nonblocking rabbit serum. E, BrdU+nuclei in the hippocampus of an exercised animal receiving a simultaneous infusion of anti-IGF-I blocking antibody. Note that the number of BrdU nuclei is considerably lower after anti-IGF-I treatment. In both D and E, image contrast is sharp to delineate easily the granule cell layer. F,Identification of BrdU-positive nuclei (green) as neurons using double fluorescence immunohistochemistry for β-tubulin III (red cytoplasm). Cells shown are located in the inner part of the granule layer. G, Whereas several GFAP-positive astrocytes (green) appeared in the dentate hilus with BrdU-positive nuclei (red), no GFAP-positive astrocytes appeared inside the granule layer.G, Granule cell layer; H, dentate hilus.Arrows indicate positive cells. H–L,Serial confocal microscopy images along the z-axis of the same neuron showing colocalization of BrdU (yellow–green patches) in the nucleus and β-tubulin (red) in the cytoplasm to demonstrate the specificity of both signals.