Mouse anti gfap

Coronal paraffin sections (8 µm) were blocked with 2% BSA and 0.1% saponin, incubated for 2 hours with primary antibodies rabbit anti-Iba1 (1∶200) (Wako Chemicals, Osaka, Japan) and mouse anti-GFAP (1∶100) (Acris antibodies) followed by incubation with secondary antibodies goat anti-rabbit AF594 and goat anti-mouse AF488 (both Invitrogen). Nuclei were counterstained with DAPI (Invitrogen) and mounted with FluoroSave reagent (Calbiochem). Fluorescent images were captured with an AxioCam MRm (Carl Zeiss, Sliedrecht, The Netherlands) on an Axio Observer Microscope with Axiovision Rel 4.6 (Carl Zeiss).

Briefly, PFA-fixed mNSCs from the co-culture assay were blocked with 5% BSA and 0.1% saponin for 30 minutes followed by incubation for 1 hour at room temperature with primary antibodies: mouse anti-nestin (1∶200) (BD Biosciences, Breda, The Netherlands), rabbit anti-Olig2 (1∶400) (Millipore), mouse anti-GFAP (1∶100) (Acris antibodies, Herford, Germany) or rabbit-anti βIII-Tubulin (1∶1000) (Abcam antibodies, Cambridge, UK). Secondary antibodies goat anti-mouse AF488 or goat anti-rabbit AF594 (Invitrogen, Paisley, UK) were incubated for one hour at room temperature. Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Invitrogen) and mounted with FluoroSave reagent (Calbiochem, Nottingham, UK). Fluorescent images were taken with an AxioCam MRm (Carl Zeiss, Sliedrecht, The Netherlands) on an Axio Observer Microscope with Axiovision Rel 4.6 software (Carl Zeiss).