Chick anti gfap

The cultured cells were planted on coverslips and fixed in 4% formaldehyde for 20 min at RT and then washed with PBS for three times. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min, then blocked with 2% BSA in PBS for 1 h at RT. Subsequently, they were incubated overnight at 4 °C with primary antibodies including rabbit anti-MAP 2 (1:1000; Millipore), mouse anti-βIII-Tubulin (Tuj1) (1:500; sigma) and chick anti-GFAP (1:500; Millipore). Coverslips were washed and incubated for 1 h at RT with secondary antibodies including anti-rabbit IgG (coupled with Alexa Fluor 568, Life Technologies), anti-rabbit IgG (coupled with Alexa Fluor 488, Life Technologies) and anti-chicken IgG (coupled with Alexa Fluor 488, Life Technologies), anti-mouse IgG (coupled with Alexa Fluor 488, Life Technologies). Nuclear DNA was stained with DAPI. Then the coverslips were mounted on glass slides with mounting buffer (Sigma-Aldrich). Immunostaining was examined by a Zeiss 710 confocal laser scanning microscope.

Antibodies used in this study are in the followings: mouse anti-E-cad (BD Transduction Laboratories 61081, 1:250); chick anti-GFAP (Millipore, 1:5,000); chick anti-GFP IgY (Abcam ab13970, 1:5,000); rabbit anti-GLAST polyclonal antibody (Covalab (France) PA-INS, 1:1,000); rabbit anti-Ki67 polyclonal antibody (Novocastra, NCL-Ki67p, 1:1,000); goat anti-NeuroD antibody (N-19; Santa Cruz, sc-1,084, 1:1,000); rabbit anti-Olig2 IgG (Millipore, AB9610, 1:1,000); goat anti-Olig2 (R&D systems, 1:1,000); mouse anti-PCNA (Novocastra, PC-10, 1:100); rabbit anti-PSmad3(S423/S425) polyclonal antibody (Millipore 07-1389, 1:1,000); mouse anti-phospho-vimentin IgG (MBL, D076-3S, 1:1,000); goat anti-Prox1 IgG (R&D systems, 1:1,000); mouse anti-S100β (Sigma, 1:1,000); rabbit Sox2 antibody (Millipore, 1:1,000); goat anti-Sox2 IgG (R&D systems, 1:1,000); goat anti-Sox9 polyclonal antibody (R&D systems, 1:1,000).