Stained Oli-Neu cells were photographed with a fluorescence microscope (Eclipse Hoechst staining TE 2000-S microscope; Nikon, Tokyo, Japan) while for AGC1+/+ and AGC1+/− neurospheres and brain section, images were acquired by using a Nikon EZ-C1 confocal microscope with a 10X or 40X objective by using the z-stack function and setting 512 steps at a stack thickness of 1 μm (40 total stacks). After image acquisition, 3D image reconstruction was performed by using the z-project plugin in Fiji ImageJ2 software (Fiji, RRID:SCR_002285) and selecting the sum function.
For Tissue section images, AGC1+/+ and AGC1+/− matched slice images were selected considering always the same rectangular area of corpus callosum (CC) and subventricular zone (SVZ) (400 µm × 161 µm). Cell volume was determined by considering a 40 µm thickness for brain sections under study and stained cell number was expressed as cells/ µm3. All positive cells were counted by using the manual cell counter plugin of Fiji ImageJ2 software (Fiji, RRID:SCR_002285). Labelling index was expressed as the ratio of positive/total cells; total nuclei were stained with Hoechst.
For Tissue section images, AGC1+/+ and AGC1+/− matched slice images were selected considering always the same rectangular area of corpus callosum (CC) and subventricular zone (SVZ) (400 µm × 161 µm). Cell volume was determined by considering a 40 µm thickness for brain sections under study and stained cell number was expressed as cells/ µm3. All positive cells were counted by using the manual cell counter plugin of Fiji ImageJ2 software (Fiji, RRID:SCR_002285). Labelling index was expressed as the ratio of positive/total cells; total nuclei were stained with Hoechst.