Chicken anti β 3 tubulin

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).

Accustain® fixed neuronal cell cultures were washed 3× 10 minutes in phosphate buffered saline (PBS), blocked using a blocking solution (BS) (0.2% Triton X-100 in PBS and 5% donkey serum (DS)) for 1 hour at RT, and incubated with mouse anti-TH (1:500, Millipore), chicken anti-βIII-tubulin (1:500, Millipore) and rabbit anti-TOM20 (1:200, FL-145, Santa Cruz Biotechnology) or rabbit anti-NeuN (1:500, Millipore) primary antibodies in BS overnight at 4°C. On the next day cells were washed 4× 10 minutes with PBS, incubated in donkey Alexa® 488 anti-rabbit, donkey Alexa® 555 anti-mouse (Life Technologies) and donkey Alexa® 647-anti-chicken (Jackson Immunoresearch) secondary antibodies for 1 hour at RT, washed 4×10 minutes with PBS, incubated with Hoechst33342 for 10 minutes and washed once more in PBS.