Anti nestin antibody

All the neurospheres were first observed under a light microscope (IX71, Olympus
Corporation, Tokyo, Japan) for a morphological examination. Immunofluorescence was used
for identification. After several passages, purified neurospheres were allowed to adhere
to a poly-L-ornithine-coated confocal dish in the cell incubator overnight. Then, the
cells were prefixed with 4% PFA at room temperature for 15–20 min. After several washes in
PBS, the neurospheres were treated as previously described. The primary antibodies used in
this procedure were as follows: rabbit polyclonal anti-Sox2 antibody (1:200, Invitrogen)
and mouse monoclonal anti-Nestin antibody (1:100, Abcam). Goat anti-rabbit IgG (Alexa
Fluor 555, Invitrogen) and goat anti-mouse IgG (Alexa Fluor 488, Invitrogen) were used as
secondary antibodies.

Samples were initially fixed with 4% paraformaldehyde for 10 min at room temperature. Then, samples were processed with 0.1% Triton X for 5 mins, followed by 5% Bovine serum albumin blocking for 1 h at room temperature. After staining with primary antibody overnight at 4 °C (Anti-Ascl1 antibodies, 1:100, Thermo Fisher Scientific, 14579482; anti-CD31 antibody, 1:100, BD Biosciences, 565629; anti-NeuN antibody, 1:200, Millipore, MAB377; anti-doublecortin antibody, 1:100, Cell Signaling Technology, 4604; anti-Nestin antibody, 1:100, Abcam, ab11306; anti-GFAP antibody, 1:200, Innovative Research, 13-0300; anti-MAP2 antibody, 1:100, Abcam, ab32454; anti-TUBB3 antibody, 1:100, BioLegend, 801209; anti-GFP antibody, 1:100, Santa Cruz Biotechnology, sc-9996; anti-Alix antibody, 1:100, Cell Signaling Technology, 2171; anti-PSA-NCAM, 1:100, Thermo Fisher Scientific, 14911882; anti-SOX2 antibody, 1:200, Millipore, AB5603; anti-PAX6 antibody, 1:200, Thermo Fisher Scientific, 13B10-1A10; anti-Ki67 antibody, 1:200, Abcam, ab15580). After washing off the primary antibody with PBS, the samples were then incubated with secondary antibodies (1:200; Jackson Immunoresearch Laboratories) for 1 h at room temperature. After that, the samples were covered with VECTASHIELD with DAPI (Vector Laboratories). Immunostaining was analyzed with a fluorescence microscope (Nikon).

Cells were fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature and then permeabilized using blocking solution (10% (v/v) horse serum in PBS with 0.1% Tween 20 (0.1% PBST)) for 60 minutes at room temperature. Cells were then incubated with the anti-Nestin antibody (Abcam, Cambridge, England, Ab5968; 1/800 in blocking solution) overnight at 4°C. After washing in 0.1% PBST, cells were incubated with the anti-rabbit Cy2 secondary antibody (Jackson, Pennsylvania, USA, 711-225-152; 1/200 in blocking solution) for two hours at room temperature. After washing in 0.1% PBST, cells were then incubated in 300 nM 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes at room temperature after which they were washed twice in 0.1% PBST and viewed under fluorescence microscopy. For quantification of Nestin expression, ImageJ was used to calculate the Nestin:DAPI ratio. For each field, the overall fluorescence intensity was calculated for each color channel and from this the ratio of Nestin:DAPI fluorescence was determined.