Identifying Neurogenesis in Traumatic Brain Injury

Newly generated neurons in the DG were identified by double labeling for BrdU and NeuN after TBI [43] (link). Briefly, after being deparaffinized and rehydrated, tissue sections were boiled in 10 mM citric acid buffer (pH 6) for 10 min. After washing with PBS, sections were incubated in 2.4 N HCl at 37°C for 20 min. Sections were incubated with 1% BSA containing 0.3% Triton-X-100 in PBS. Sections were then incubated with mouse anti-NeuN antibody (1∶200; Chemicon, Temecula, CA) at 4°C overnight. For negative controls, primary antibodies were omitted. FITC-conjugated anti-mouse antibody (1∶400; Jackson ImmunoResearch, West Grove, PA) was added to sections at room temperature for 2 h. Sections were then incubated with rat anti-BrdU antibody (1∶200; Dako, Glostrup, Denmark) at 4°C overnight. Sections were then incubated with Cy3-conjugated anti-rat antibody (1∶400; Jackson ImmunoResearch, West Grove, PA) at room temperature for 2 h. Each of the steps was followed by three 5-min rinses in PBS. Tissue sections were mounted with Vectashield mounting medium (Vector laboratories, Burlingame, CA).