Lsm 710 microscope system

In order to visualize the neocortical layers, the embryonic brain sections were stained with TO-PRO-3 following the manufacturer's instructions (Invitrogen) and mounted in Vectashield with DAPI (Vector Laboratories) before being imaged via confocal microscopy. For each sample, two z-stack images at the level of the future somatosensory cortex in each hemisphere were acquired with a Zeiss LSM 710 microscope system using Zen software. Using LSM Image Browser software (Zeiss), the thickness of the VZ and SVZ, the IZ, the CP, and total pallial thickness for each image, was measured. The measurements were averaged for each sample and normalized to the mean of euploid littermates. Measurements were then averaged by genotype and compared using an unpaired two-tailed Student's t-test.

Immunofluorescence staining of differentiated cells was performed as previously reported [2 (link)]: The primary antibodies used were anti-βIII-tubulin mouse monoclonal (1:500, MMS-435P, Tuj1 clone, Covance, Princeton, NJ, USA); anti-tyrosine-hydroxylase (TH) rabbit polyclonal (1:100, NB300-109, Novus Biologicals, Centennial, CO, USA); anti-dopamine polyclonal antibody (1:250, IS1005, ImmuSmol, Pessac, France) with a STAINperfect immnostaining kit A (SP A-1000, ImmuSmol); anti-dopamine-transporter (DAT) rabbit monoclonal antibody (1:250, ab184451, Abcam, Cambridge, UK); and anti-Nurr1 rabbit polyclonal (1:50, 10975-2-AP, Proteintech, Rosemont, IL, USA). The secondary antibodies were Alexa Fluor^® 568-conjugated goat anti-mouse (1:400, A11004, Molecular Probes, Eugene, OR, USA); Alexa Fluor^® 488-conjugated goat anti-rabbit (1:400, A11008, Molecular Probes); and Alexa Fluor^® 568-conjugated goat anti-rabbit (1:400, A21069, Molecular Probes). Counterstaining was performed with 4′, 6-diamino-2-phenylindole, dihydrochloride (DAPI) (SE196, DOJINDO, Kumamoto, Japan). Images were collected using an LSM 710 microscope System with ZEN software (Carl Zeiss, Oberkochen, Germany).