Tubb3 tuj1

Neural cultures were grown on Matrigel-coated plastic coverslips in 3 N medium and were fixed in 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized in 0.01% Triton X-100 (Ajax Finechem) in PBS for 15 min at room temperature. All cells were blocked for 1 h with 10% goat serum (Invitrogen) in PBS. Primary antibodies used were OCT4 (1:100; Millipore), NANOG (1:100; Millipore), CUX1 (1:100; Abcam), glial fibrillary acidic protein (GFAP) (1:250; Dako), TUBB3/TUJ1 (1:1,000; Covance), BRN2 (1:100; Abcam), PAX6 (1:1,000; Developmental Studies Hybridoma Bank [DSHB]), anti-phospho-histone H3 (Ser10) (1:200; Cell Signaling Technology) and were applied for 3 to 4 h at room temperature or overnight at 4°C. Isotype- and species-matched Alexa Fluor-conjugated secondary antibodies (1:1,000; Invitrogen) were applied for 1 h at room temperature. Cells were washed in PBS and mounted on glass slides with ProLong Gold antifade containing 4′,6′-diamidino-2-phenylindole (DAPI; Invitrogen) and imaged using an Olympus IX51 (Olympus) fluorescence microscope equipped with a MicroPublisher, version 3.3, real-time viewing (RTV) charge-coupled-device (CCD) camera (QImaging) using Q-Capture Pro, version 6.0, software.

Cells were fixed in 3.8% (w/v) formaldehyde for 10 min at room temperature, and blocked with 10% (v/v) FBS in PBS containing 0.1% (v/v) Triton X-100 for 1 h at room temperature. Cells were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were: anti-neuron-specific class III beta-tubulin (TUBB3, TUJ1, 1:7500, Covance Inc. Princeton, NJ, USA), microtubule-associated protein 2 (MAP2; 1:500, Millipore, Bedford, MA, USA), SMI32 (1:1000, Covance Inc. Princeton, NJ, USA), ISL1/2 (39.4D5, 1:5, developed by T.M. Jessell and S. Brenner-Morton was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242), S100β (1:200) and vimentin (1:1000 Progen, Heidelberg, Germany). The following day, coverslips were washed and incubated with Alexa-Fluor conjugated secondary antibodies (1:1000) for 1 h at room temperature, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 0.3 µM). Cells were mounted in Aqua-Polymount (Polysciences, Inc., Warrington, PA, USA).