Ab144p

For immunocytochemistry and immunohistochemistry, samples were blocked in 10% normal goat serum (NGS) or 10% normal donkey serum (NDS) as appropriate and permeabilised in 0.3% Triton X-100 (Sigma-Aldrich; in PBS) at room temperature (RT) for 1 h. Immunolabelling was performed with primary antibodies in NGS (5%) and Triton X-100 (0.1% in PBS) at 4 °C overnight followed by species-specific secondary antibodies for 1 h at RT and DAPI nuclear counterstain (100 ng/ml) for 10 min at RT. For human post-mortem samples fixation and permeabilisation in cold methanol (−20 °C, 20 min) was performed before the immunostaining. Primary antibodies were diluted as follows: rabbit anti Olig2 (Millipore, AB9610) 1:200; mouse anti SMI32 (Cambridge Bioscience, SMI-32R-500) 1:1000; goat anti ChAT (Millipore, AB144P) 1:100; rabbit anti Pax6 (biolegend, 901301) 1:300; mouse and rabbit anti SFPQ (Abcam, ab11825 and ab38148, respectively) 1:100; mouse and rabbit anti beta-tubulinIII (biolegend, 801201), mouse anti Lim3 (DSHB, 67.4E12) 1:50; chicken anti Nkx2.2 (DSHB, 74.5A5) 1:50. Images were acquired using either a 710 Laser Scanning Confocal Microscope (Zeiss) or the Opera Phenix High-Content Screening System (Perkin Elmer). For image analysis, Fiji or the Columbus Image Analysis System (Perkin Elmer) were used.