To investigate BrdU-positive (BrdU+) cell phenotype, triple fluorescence immunohistochemistry for a neuron specific marker (neuronal nuclei: NeuN), astroglia specific marker (glial fibrillary acidic protein: GFAP), and BrdU was conducted on the 4D+28 tissue. Identical to procedures reported previously, every 12th section had DNA denatured before blocking in TBS+ (Nixon and Crews, 2004; Nixon et al., 2008). The sections were then incubated for 48 h at 4°C in the following primary antibodies in TBS+: rat anti-BrdU (1:400, Accurate, Westbury, NY), mouse anti-NeuN (1:500, Chemicon) and rabbit anti-GFAP (1:2500, DAKO, Glostrup, Denmark). Following rinses in TBS+, the sections were incubated for 1 h in fluorescent-conjugated secondary antibodies: goat anti-rat Alexa Fluor 488, goat anti-mouse Alexa Fluor 633, and goat anti-rabbit Alexa Fluor 555 (1:200, Molecular Probes, Invitrogen, San Diego, CA). Following washes in TBS, the sections were mounted on glass slides and coverslipped, using Pro-long® Gold anti-fade reagent (Molecular Probes, Invitrogen). Sections were kept in the dark at all points possible from secondary incubation forward.
