Dissected brains were fixed in 4% paraformaldehyde overnight, following dehydration in 25% sucrose in phosphate-buffered saline (PBS). All sections for immunohistochemistry were sliced at a thickness of 40 µm. Brain sections were mounted on SuperFrost Plus slides (Thermo) and dried overnight. Slides were then incubated in 0.01 M citric acid buffer for 20 min at 95 °C, 3% H2O2 for 10 min, rinsed in PBS, and incubated overnight at room temperature in DCX antibody (1:250, Santa Cruz), Ki67 antibody (1:1,000, Vector Laboratory), Tbr2 antibody (1:1,000, Abcam), and NeuroD (1:1,000, Santa Cruz). Subsequently, we used a standard IgG ABC kit (Vector Laboratory) procedure according to the manufacturer’s instructions and incubated the slides for 5 to 10 min with a Sigma DAB (3,3′-diaminobenzidine) tablet.
For quantification, all slides were randomized and coded before quantitative analysis. Slides (half-brain) were examined under a 20× objective. Labeled cells were counted on every eighth section through the entire rostrocaudal extent of the granule cell layer (six sections per animal). The number of cells counted was then multiplied by 16 to obtain an estimate of the total number of positive cells in the dentate gyrus, as previously described (37 (link)).
For quantification, all slides were randomized and coded before quantitative analysis. Slides (half-brain) were examined under a 20× objective. Labeled cells were counted on every eighth section through the entire rostrocaudal extent of the granule cell layer (six sections per animal). The number of cells counted was then multiplied by 16 to obtain an estimate of the total number of positive cells in the dentate gyrus, as previously described (37 (link)).