Immunostaining and Imaging of Neurospheres

Neurospheres were fixed in 4% (w/v) paraformaldehyde (PFA) + 4% (w/v) sucrose in PBS for 20 min and processed for immunostaining as previously described43 (link). Primary and secondary antibodies were used as follows: mouse anti-βIII-tubulin (1:200; Millipore Darmstadt, Germany, MAB1637); rabbit anti-GFAP (1:200; Millipore, AB5804); AlexaFluor^® 488 goat anti-mouse IgG (1:500; Life Technologies, A11001) and AlexaFluor^® 594 goat anti-rabbit IgG (1:500; Life Technologies, A11012). Cell nuclei were counterstained with TO-PRO-3 (Life Technologies). Samples were visualized using point-scan confocal microscopy (SP5, Leica, Wetzlar, Germany) or light-sheet microscopy44 (link)45 (link). Merge between channels and maximum z-projections, as well as linear brightness and contrast adjustments of the images, were performed using the open source FIJI software46 (link).