Triple staining was performed to identify the phenotype of the new cells (Fig 1 ).
Sections were processed following the immunohistochemistry protocol described above. Samples were incubated in primary antibody rat anti BrdU (1:800, AbD Serotec) for at least16 h at 4°C followed by incubation for 2 h with the secondary antibody rat IgG (1:500, Vector). After incubation, the samples were placed in ABC and revealed with tyramide signal amplification (TSA) plus coumarin system (1:100, PerkinElmer) according to vendor’s instructions. Sections were rinsed and incubated in mouse monoclonal anti- neuronal nuclei antibody (NeuN) to identify mature neurons (1:250, Millipore) for 20 h at 4°C and incubated in mouse IgG (1:300, Vector). Later, samples were incubated in ABC complex and revealed using TSA plus cyanine 3 (CY3) system (1:100). Sections were rinsed and incubated with primary antibody rabbit monoclonal anti- glial fibrillary acidic protein to identify glial cells (GFAP, 1:100, AbD Serotec) and incubated with secondary anti-rabbit IgG Alexa Fluor 488 (Life science, 1:1250). Finally, brain sections were mounted on slides and cover slipped using aqua poly/mount (Polysciences).
Sections were processed following the immunohistochemistry protocol described above. Samples were incubated in primary antibody rat anti BrdU (1:800, AbD Serotec) for at least16 h at 4°C followed by incubation for 2 h with the secondary antibody rat IgG (1:500, Vector). After incubation, the samples were placed in ABC and revealed with tyramide signal amplification (TSA) plus coumarin system (1:100, PerkinElmer) according to vendor’s instructions. Sections were rinsed and incubated in mouse monoclonal anti- neuronal nuclei antibody (NeuN) to identify mature neurons (1:250, Millipore) for 20 h at 4°C and incubated in mouse IgG (1:300, Vector). Later, samples were incubated in ABC complex and revealed using TSA plus cyanine 3 (CY3) system (1:100). Sections were rinsed and incubated with primary antibody rabbit monoclonal anti- glial fibrillary acidic protein to identify glial cells (GFAP, 1:100, AbD Serotec) and incubated with secondary anti-rabbit IgG Alexa Fluor 488 (Life science, 1:1250). Finally, brain sections were mounted on slides and cover slipped using aqua poly/mount (Polysciences).