Beta 3 tubulin

Briefly, HS420 hESC and ReNcells cultured on cover slips coated with Matrigel (1/30 in DMEM) were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min at room temperature, washed and processed for conventional immunocytochemistry. Cells were incubated with a primary antibody Oct4 (rabbit polyclonal 1/200, Abcam, ab18976), Nanog (mouse monoclonal, 1/800, Abcam ab 171380), Sox2 (goat polyclonal 1/400, ThermoFisher, tf-pa5-18406,), Beta-III tubulin (mouse monoclonal, 1/1000, Sigma, T8660), γ-H2A.X (rabbit polyclonal 1/1000, Abcam ab11174), Ki67 (rabbit monoclonal 1/250 Abcam, ab16667) diluted in PBS-Triton 0.1% for 1 h at room temperature (RT). Detection of primary antibodies was performed using appropriate species-specific Alexa 488-, Alexa 555- or Alexa 647-labeled secondary antibodies for 1 h at RT in the dark. The cells were finally stained with Hoechst 33342 or 4′-6-diamidino-2-phenylindole (DAPI) for nuclei identification. Slides were mounted in Fluorosave (Calbiochem, Merck Millipore). Controls included examination of the cell or tissue autofluorescence and omission of the first antibody. Images of the immunostained cells were captured on a Zeiss Axioskop 2 fluorescence microscope with an Axiocam Color HRC detector (Zeiss, Feldbach, Switzerland).

Animals were first sacrificed with an overdose of sodium pentobarbital, and perfused transcardially with PBS followed by 4% PFA, before dissecting out the spinal cord and sciatic nerve. The tissue was cryoprotected in 30% sucrose solution at 4 °C for 3 days, embedded in OCT (RALamb UK) and cut longitudinally using a cryostat into 14 μm sections. Sections were washed in PBS (15 min, room temperature), permeabilized with 0.1% Triton X-100 (15 min, room temperature), blocked with 10% goat serum (1 h, room temperature) and incubated in primary antibodies (diluted in blocking solution) at 4 °C overnight. The following day, sections were washed with PBS, incubated with secondary antibodies (1 h, room temperature) and then mounted in Fluorosave. Primary antibodies were used against: talin (clone 8D4, Abcam, 1:20) and beta III tubulin (Sigma, 1:400).

Neurones were cultured on laminin‐coated 1,5 mm cover‐slips and fixed for 20 min using 3% paraformaldehyde (VWR) at room temperature followed by permeabilization and blocking, using 0.1% saponin and 1% BSA (Sigma‐Aldrich) in PBS for 1 hr. Immunostaining with beta‐III Tubulin (#T2200, Sigma‐Aldrich) was performed overnight, while samples were incubated with the secondary antibody Alexa Fluor® 488 (Thermo Fisher Scientific) for 1 hr at a 1:1,000 dilution. Slides were mounted with ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific) and 0.01 μg/ml DAPI (Sigma‐Aldrich). A Carl Zeiss fluorescent microscope was used for image acquisition.