Rabbit anti cow gfap

Immunohistochemistry was performed in the same manner as described by Boutin et al. [31 (link)]. Sequential sections of tissue, adjacent to those used for autoradiography, were stained either for glial fibrillary acidic protein (GFAP) and CD11b antigen (Ox42) or for GFAP and neuron-specific nuclear protein (NeuN). Sections were fixed in 10% formalin (Sigma-Aldrich, USA) for 1 h, washed in 6 × 5 min PBS, and then incubated for 20 min in diluent (PBS with 5% normal horse serum and 0.1% Triton X-100). Sections were then incubated overnight at 4°C with primary antibodies in diluent (rabbit anti-cow GFAP, 1 : 1,000, DakoCytomation; mouse anti-rat CD11b, 1 : 1,000, Serotec; mouse anti-mouse NeuN, 1 : 1,000, Chemicon) and then washed in 3 × 5 min PBS. Incubation in secondary antibodies was for 1 h at RT (Alexa Fluor 594 nm goat anti-mouse IgG, 1 : 500; Alexa Fluor 488 nm goat anti-rabbit IgG, 1 : 500 (Molecular Probes, Invitrogen)) and then sections were washed again in 3 × 5 min PBS. Sections were mounted with an antiadherent solution (Citifluor, UK). Sections incubated without primary antibodies served as negative controls.
Immunofluorescence images were obtained with an Olympus BX-41 microscope equipped with a digital camera (QImaging, Canada) at 10x magnification, acquired with Image-Pro software. Image fusion was performed with MCID image analysis software.

Cells were fixed in cold 4% paraformaldehyde in PBS for 10 min. iPSCs, NPCs and neurons were permeabilized at room temperature for 15 min in 0.2% TritonX-100 in PBS. Samples were blocked in 5% BSA with 0.1% Tween 20 for 30 min at room temperature. The primary antibodies and dilutions used were: goat anti-SOX2 (Santa Cruz), 1:200; mouse anti-human Nestin (Chemicon), 1:200; rabbit anti-βIII-tubulin (Covance), 1:200; mouse anti-βIII-tubulin (Covance), 1:200; rabbit anti-cow-GFAP (Dako) 1:200; mouse anti-MAP2AB (Sigma), 1:200; secondary antibodies were Alexa donkey 488 and 568 anti-mouse, rabbit and goat (Invitrogen), used at 1:1000. Nuclear stainings were done with Hoechst (Invitrogen).