Mouse monoclonal anti brdu

Brain removal, processing, sectioning, and immunohistochemical detection of BrdU and other cell markers were performed as previously described^{28 (link),29 (link)}. Primary antibodies used were: mouse monoclonal anti-BrdU (1:100) and rabbit polyclonal anti-GFAP (1:3000) from Dako (Glostrup, Denmark); rat monoclonal anti-BrdU (1:100) from Abcam (Cambridge, UK); goat polyclonal anti-doublecortin (1:200) and goat polyclonal anti-nestin (1:200) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-NeuN (1:100), goat polyclonal anti-ChAT (1:100), and mouse monoclonal anti-parvalbumin (1:100) from Merk Millipore (Billerica, MA, USA), and mouse monoclonal anti-VGLUT2 (1:100) from Abcam (Cambridge, UK). Secondary fluorescent antibodies used (1:1000) were all from Invitrogen (Carlsbad, CA, USA): donkey anti-rabbit IgG conjugated to AlexaFluor 594 or AlexaFluor 488; donkey anti-mouse IgG conjugated to AlexaFluor 405 or AlexaFluor 488; donkey anti-goat conjugated to AlexaFluor 594; and donkey anti-rat conjugated to AlexaFluor 488 or AlexaFluor 594. Biotinylated donkey anti-goat IgG (1:250) was from Sigma-Aldrich.

Additional tissues were harvested, cultured and labeled according to the following steps. Lectin + NG2: 1) 1:100 rabbit polyclonal NG2 antibody (Millipore; Billerica, MA); 2) 1:100 goat anti-rabbit CY3-conjugated antibody (GAR-CY3) and 5% NGS. Lectin + BrdU: 1) 2 h incubation in 1mg/ml BrdU (Sigma-Aldrich; St. Louis, MO) supplemented media; 2) 6 M HCl for 1 h at 37°C to denature the DNA; 3) 1:100 mouse monoclonal anti-BrdU (Dako; Carpinteria, CA); 4) 1:100 GAM-CY3 Fab fragments (Jackson ImmunoResearch Laboratories; West Grove, PA). Live/Dead: 1) 1:400 Calcein AM and 1:200 EthD-1 (Molecular Probes; Grand Island, NY) for 10 min at 37°C. Lectin labeling was performed as described above. Tissues were fixed with methanol fixation at -20°C for 30 min. All antibodies were diluted in antibody buffer solution (PBS + 0.1% saponin + 2% bovine serum albumin).

Brain processing and immunohistochemical detection of the proliferation marker BrdU, the astrocyte and neural stem cell marker GFAP, the neural progenitor cell marker nestin, the early neuronal differentiation marker doublecortin (DCX), and the mature neuron maker NeuN were performed as previously described^{49 (link)}.
Primary antibodies used were mouse monoclonal anti-BrdU (1:100) from Dako (Hamburg, Germany) or rat monoclonal anti-BrdU (1:100) from Abcam (Cambridge, UK), mouse polyclonal anti-GFAP both from Cell Signaling (Beverly, MA, USA), goat polyclonal anti-DCX (1:500), goat polyclonal anti-nestin (1:500), and mouse monoclonal anti-NeuN (1:100) all of them from Abcam (Cambridge, UK); goat anti-ChAT, polyclonal, 1:100, Merk Millipore (Billerica, MA, USA) mouse anti-parvalbumin, monoclonal, 1:100, Merk Millipore (Billerica, Ma, USA). Secondary antibodies used were Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-mouse, Alexa Fluor 405 goat anti-mouse, Alexa Fluor 594 donkey anti-rat, Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 594 donkey anti-rabbit and Alexa Fluor 594 donkey anti-goat (all at 1:1000, from Life Tech).