Als had been visualized using the VIP substrate kit (Vector Labs) using the manufacturer’s protocol. Sections were counterstained with 0.1 methyl green (wt/vol), dehydrated, and mounted in DPX (Fluka). Antibodies Main antibodies are listed in Supplementary Table 2. Secondary antibodies utilized: FITCand Cy3-conjugated secondary antibodies (Jackson Immunologicals), Alexa Fluor 350-, 488- and 568-conjugated goat anti-mouse, Alexa Fluor 488- and 568-conjugated goat antirabbit, Alexa Fluor Chlorobutanol Autophagy 488-conjugated goat anti-guinea pig and Alexa Fluor 568-conjugated anti-human antibodies (Life technologies). MicroscopyAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptBrain histological sections have been imaged using a macroscope (Olympus MVX10) and neocortical thickness was Methylisothiazolinone Autophagy measured in comparative sections in the pia towards the white matter. At P2 cortical thickness was measured in motor, somatosensory and visual cortex (two.31mm, three.75mmand 4.23mm, respectively according to reference66) and at P60 in motor and visual cortex (Bregma: 1.42 and -2.70, respectively). Fluorescent images were acquired with an Orca AG camera (Hamamatsu) mounted on a Leica DMI6000B microscope equipped with 1.four NA 63and 100oil immersion objectives. AF6000 application (Leica) was applied for image acquisition and deconvolution of z-stack pictures (distance between z-slices was 0.2 ). Coronal/sagittal serial brain sections from embryos at E14.5 from multiple animals per genotype were analyzed in every experiment (particulars in figure legends). For cell counts in the cortex all cells from the ventricular surface for the pial surface have been counted and normalized using the region chosen (mm2). Extra image processing and maximum intensity zprojections had been carried out in ImageJ computer software. Mitotic figure classification was done employing apical mitosis within cortical sections imaged with a Leica TCS SP5 laser scanning spectral confocal microscope set up on a Leica DMI600 inverted microscope. Confocal Z-stacks had been acquired with 0.2 of step size and employing laser parameters which minimized the presence of saturated pixels. -tubulin distribution in mitotic centrosomes was calculated assigning centrosomes to the “2 bright poles” category when each -tubulin signals fell inside the 400 of distribution of their bright/dim ratio and “1 dim or absent pole”Nat Commun. Author manuscript; readily available in PMC 2016 January 09.Marjanovi et al.Pagecategory if one of the -tubulin signals fell within a 6000 distribution of bright/dim ratio. Transmission electron microscopy Testes of mature mice had been dissected and fixed for transmission electron microscopy (TEM) evaluation as previously described67. Briefly, testes have been pre-fixed in cacodylate-buffered glutaraldehyde and post fixed in buffered OsO4 followed by RenlamM-1 resin (Serva, Heidelberg, Germany). Ultrathin sections were analyzed in a Tecnai12 BioTwin TEM (FEI, Eindhoven, NL) and imaged having a CCD camera (SIS MegaView3, Surface Imaging Systems, Herzogenrath, Germany) along with the Analysis Imaging Interface. Contrast and brightness of pictures have been additional adjusted working with Adobe Photoshop CS. StatisticsAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptAll graphics with error bars are presented as average + s.d. (except in Fig. 6c where only average bar is shown and Fig. 1a-1b and 5b where median with 1st and 3rd quartile from the box plot is shown. To determine statistical significance between samples, unpaired two-way Wilcoxon rank-sum test was use.