Immunostaining Procedure

For immunostaining, the following primary antibodies were used: rat monoclonal antibody to GFP (B-Bridge, clone 1A, 1:1000), rabbit polyclonal antibody to GFP (Life Technologies, A6455, 1:200), rabbit polyclonal antibody to Ki67 (AbCam, ab15580, 1:250), mouse monoclonal antibody to phospho-Histone H3 (Thermo Fisher, MA5–15220, PH3, 1:500), goat polyclonal antibody to Sox2 (Santa Cruz, sc-17320, 1:250), rabbit polyclonal antibody to Tbr2 (AbCam, ab23345, 1:500), chicken polyclonal antibody to Map2 (Abcam, ab5392, 1:5000), rabbit polyclonal antibody to pericentrin (Abcam, ab4448, 1:200) or revelation kit for EdU detection (Thermo Fisher). The secondary antibodies used were: RRX-, FITC- or Cy5-conjugated antibodies to rabbit, mouse, goat or rat IgG (Jackson ImmunoResearch, 1:500). Biocytin and Rhodamine Avidin DCS were purchased from Invitrogen (B1592) and Vector Lab (A-2012), respectively.

For IHC, cryostat sections were washed one time with PBS and incubated 1 h at room temperature in blocking solution (0.3% TritonX-100, 10% normal donkey serum (Jackson ImmunoResearch) in PBS). Slides were incubated overnight at 4°C with primary antibodies and for 1 h at room temperature with secondary antibodies all diluted in blocking solution. For EdU immunostaining, after incubation with the secondary antibody, slices were incubated during 30 min with the appropriate preparation of EdU detection mix according to the protocol of kit (Thermo Fisher). Slides were washed again with PBS and mounted in Vectashield Hard Set Mounting Medium with DAPI. Images were captured using an Olympus Fluoview FV1000 confocal system equipped with an Olympus IX81 inverted microscope (Olympus or Nikon).