Cy3 conjugated donkey anti rat

For quantification of specific GFP positive cell populations, the following triple labeling was performed: mature neurons—mouse anti-GFP (1:200; Santa Cruz Biotechnology), chicken anti-β-III tubulin (1:100; Millipore), and rat anti-BrdU (1:400; Accurate Chemical & Scientific Corp.); neuroblasts—mouse anti-GFP, goat anti-doublecortin (DCX; 1:400; Santa Cruz Biotechnology), and rat anti-BrdU; progenitor cells—mouse anti-GFP, rat anti-BrdU, rabbit anti-nestin (1:200; Millipore); stem cells- goat anti-GFP, mouse anti-nestin, rabbit anti-GFAP (1:500; Dako); mature astrocytes—goat anti-GFP, mouse anti-GFAP, rabbit anti-S100ß (1:3000, Abcam). The following secondary antibodies were used from Jackson ImmunoResearch Laboratories (West Grove, PA): biotinylated species-specific anti-IgG (all used at 1:250), Cy3-conjugated Donkey anti-Rat (1:500), Alexa Fluor 647 (AF647)-conjugated Donkey anti-Mouse (1:250), Cy2-conjugated Streptavidin (1:250), DAPI Nucleic Acid Stain (1:10,000, Life Technologies, Grand Island, NY).

50 μm sagittal sections cut using a sliding freezing microtome (Leica Biosystems, Buffalo Grove, IL) were stored in cryoprotectant (glycerol, ethylene glycol, 1X PBS) at −20 °C. The following antibodies were used: rat anti-BrdU (1:400; Accurate Chemical & Scientific Corp., Westbury, NY), mouse anti-nestin (1:100; Millipore Corporation, Billerica, MA), goat anti-doublecortin (DCX; 1:400; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-NeuN (1:400, Millipore, Temecula, CA), rabbit anti-Egr-1 (1:250, Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-green fluorescent protein (GFP, 1:1000, Abcam, Inc. Cambridge, MA) and mouse anti-green fluorescent protein (GFP, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA): biotinylated species-specific anti-IgG (all used at 1:250), Cy3-conjugated Donkey anti-Rabbit (1:250), Cy3-conjugated Donkey anti-Rat (1:250), Alexa Fluor 647 (AF647)-conjugated Donkey anti-Mouse and anti-Rat (1:250), Cy2-conjugated Streptavidin (1:250), DAPI Nucleic Acid Stain (1:2500, Life Technologies, Grand Island, NY).