Anti nestin

Cells and cortical sections were fixed in 4% PFA in 0.12 M PB, pH 7.2 at 4°C for 5 min and incubated in Blocking buffer (BB; 0.12 M PB, pH 7.2, 0.15% glycine, 2 mg/ml BSA fragment V (Gibco-Invitrogen) and 0.1% Triton X-100) for 1 h on ice. Cells were incubated o/n at 4°C with primary antibodies in BB. After extensive washes with PBS at RT, cells were incubated with species specific secondary antibodies (Alexa 488-conjugated, 1:500; Invitrogen) for 1 h at RT. Samples were mounted in anti-fade DAPI mounting media (Invitrogen) and photographed on a Leica SP5 confocal microscope. Primary antibodies: anti-Nestin (mouse IgG 1:75, Developmental Studies Hybridoma Bank (DSHB), University of Iowa, http://dshb.biology.uiowa.edu), anti-RC2 (mouse IgM 1:100, DSHB), anti-GFAP, Cy3-linked (mouse IgG 1:100, SIGMA), anti-Sox2 (rabbit IgG 1:1000, Santa Cruz, INC).

To characterize differentiation, retinoic acid-treated CE1 cells were cultured on either ACMFP fibers or flat PLGA membrane (control) for 6 days as described above. Cells were fixed in 4% paraformaldehyde, blocked with donkey serum in 0.5% Triton X-100 for 30 min, incubated with primary antibodies at 4 °C overnight followed by secondary antibody treatment at room temperature for 2 hr. Primary antibodies used in this study included: Anti-Sox2 (1:200; Abcam), Anti-Nestin (1:200; Developmental Studies Hybridoma Bank), Anti-TUJ1 (1:500; AVES), Anti-GFAP (1:200; Santa Cruz Biotechnology) and Anti-MOG (1:200; Millipore) antibodies. Secondary antibodies included Dylight 549 or Dylight 649 conjugated donkey anti-mouse, rabbit or chicken antibodies (Jackson Immunoresearch). 4,6-Diamidino-2-Phenylindole (DAPI; 1:500; Invitrogen) was used to label all nuclei. Samples were observed and imaged by Leica SPE confocal microscopy and/or Leica DMI 3000B epifluorescence microscopy. Cells cultured on coverslips serve as controls. The number of Sox2 and Nestin-positive cells, TUJ1 positive cells, GFAP positive cells and MOG positive cells were quantified via the Cell Counter plugin of the ImageJ software), and (Sox2 and Nestin double-labeled cells)/(DAPI positive cells) × 100% were calculated (n = 12 samples per group, 1–2 images were randomly selected per sample).

YAC128 NSCs were transferred to 12 mm round cover slips coated with PLO/Laminin in a four‐well plate (BD Biosciences). The cells were then washed with chilled PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature. Next, the cells were permeabilized and blocked with 0.1% Triton X‐100 (Sigma‐Aldrich) and 5% normal horse serum (Vector Laboratories) diluted in PBS for 30 minutes at room temperature. The cells were then incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody. The following secondary antibodies were used: Alexa‐555‐conjugated goat anti‐mouse IgG; Alexa‐555‐conjugated goat anti‐rabbit IgG; Alexa‐488‐conjugated goat anti‐rat IgG; and Alexa‐488‐conjugated goat anti‐rabbit IgG (1:200, Molecular Probes). Images were obtained using two confocal laser‐scanning microscope systems: a Zeiss LSM 880 scanning confocal microscope (Zeiss) and a Leica TCS Sp5 II confocal microscope (Leica). The following primary antibodies were used: anti‐NESTIN (1:40, Developmental Studies Hybridoma Bank), anti‐SOX2 (1:200, Millipore) for the detection of uncommitted neural stem cells, anti‐MAP2 (1:500, Millipore), anti‐Tuj1 (1:500, Millipore) for the detection of differentiated neurons, anti‐HTT (1:100, Sigma‐Aldrich) and anti‐ubiquitin (1:50, Santa Cruz Biotechnology).