Goat polyclonal anti doublecortin dcx

The immunostaining assays were performed using a protocol adapted from^{18 (link)}. First, brain sections were incubated with 2 M HCl for 25 min at 37 °C to induce DNA denaturation. After washing with PBS, tissue sections were further incubated in a blocking solution containing 2% of horse serum (Life Technologies, Carlsbad, CA, USA) and 0.3% Triton X-100 (Fisher Scientific) diluted in 0.1 M PBS for 2 h at RT. After the blocking procedure, tissue sections were incubated for 72 h at 4 °C in the following primary antibodies (diluted in the blocking solution): rat monoclonal anti-BrdU (1:500, AbD Serotec, Raleigh, NC, USA), goat polyclonal anti-doublecortin (DCX; 1:500, Santa Cruz Biotechnology), or mouse monoclonal anti-NeuN (1:500, Merck Millipore). Then, sections were rinsed in PBS and incubated with Hoechst (1:1000; Sigma-Aldrich Co. LLC) and the respective secondary antibodies: Alexa Fluor-488 donkey anti-rat, Alexa Fluor-546 donkey anti-goat or anti-mouse (all 1:500; all Life Technologies), diluted in a solution containing 0.3% Triton X-100 in 0.1 M PBS, for 2 h at RT. Finally, sections were rinsed in PBS and mounted in Fluoroshield Mounting Medium (Abcam Plc., Cambridge, UK) for further analysis.

Cultures were fixed at the indicated time points in 4% w/v paraforlmadehyde for 20 min, washed with phosphate-buffered saline (PBS), blocked and permeabilized with 5% donkey serum in PBS containing 0.1% Triton X-100. The following primary antibodies were used: mouse monoclonal anti-PAX6 (1:50, Developmental Studies Hybridoma Bank) to identify neural progenitors; rabbit polyclonal anti-Nestin (1:200, Millipore) to identify neuroepithelial cells; goat polyclonal anti-doublecortin (DCX, 1:100; Santa Cruz) to identify neuroblasts/early neurons; rabbit polyclonal βIII-tubulin (TUJ-1 antibody 1:1000, Cell Signaling) to identify neurons; and goat polyclonal anti-Nanog (1:100, R and D) to identify pluripotent cells. After incubation with appropriate secondary antibodies conjugated with AlexaFluor 488 (green) or 546 (red) (1:500; Molecular Probes, Eugene, OR, USA), coverslips were mounted with ProLong Gold antifade reagent with DAPI (Cell Signaling). For quantification of labeled cells, images from 10 randomly selected fields from each independent experiment were obtained using a 20X lens on a Leica TCS-SP5II confocal microscope (LEICA Microsystems) and analyzed using ImageJ software (NIH).

Dulbecco’s modified Eagle’s medium (DMEM), supplemented with fetal bovine serum (FBS) and penicillin–streptomycin, was provided by Invitrogen (Carlsbad, CA, USA). Goat polyclonal anti-doublecortin (DCX) and rabbit polyclonal anti-phospho-cAMP-response-element-binding protein (CREB) (pCREB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Biotinylated horse anti-goat antibody, biotinylated goat anti-rabbit antibody, and avidin–biotin complex (ABC) were purchased from Vector Labs (Burlingame, CA, USA). Paraformaldehyde (PFA), 3,3-diaminobenzidine (DAB), sucrose, phosphate-buffered saline (PBS), 30% hydrogen peroxide (H₂O₂), lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), piracetam, dibutylphthalate polystyrene xylene (DPX) histomount medium, and mouse anti-synaptophysin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cerebrolysin was purchased from Ever Neuro Pharma (GMBH, Austria). The enzyme-linked immunosorbent assay (ELISA) development kit for NGF was purchased from R&D Systems (Minneapolis, MN, USA).