Nucspot live 650 nuclear stain

For live imaging experiments, lentiviral SYN1-GFP (PZ196, Addgene) was added to neurons in suspension following dissociation on day 14. Neurons were plated in culture media with SYN1-GFP for the first 24 hours and following a full media change were cultured as previously described. For nuclear staining, NucSpot Live 650 Nuclear Stain (Biotium) was added 1:1000 to media 1 hour prior to imaging and supplemented with media changes. Propidium iodide (Sigma) was added to media 1:2000 and supplemented with media changes. Images were collected prior to trauma and every 1.5 hours for the first 6 hours following trauma. After 6 hours, images were collected every 12 hours. Images showing synapsin-GFP alone were collected from an IncuCyte S3 (Sartorius). Images showing PI and nuclear stains were collected using a Nikon BioStation (Nikon). Image Analysis was performed using Image J (U. S. National Institutes of Health, Bethesda, Maryland, USA)

The cells were fixed in 4% paraformaldehyde and permeabilized in 0.4% to 0.5% Triton X‐100. The cells were blocked with goat/bovine serum in phosphate‐buffered saline (PBS) and incubated for 3 hours or overnight with the following antibodies, OCT4 (Abcam, Cambridge, Massachusetts; ab19857), NANOG (Abcam ab21624), TH (Abcam; ab76442), TUJ1 (Cell Signaling, Danvers, Massachusetts; 5568S), DAT (GeneTex, Irvine, California; 133152), α‐synuclein (BD Biosciences Clontech, Palo Alto, California; 610787). After several washes in PBS, the samples were incubated with Alexa Fluor‐conjugated secondary antibodies; 488‐labeled goat anti‐rabbit IgG (Invitrogen, A11008) for the detection of OCT4, 488‐labeled goat anti‐chicken IgY (Abcam; ab150169) for TH, 568‐labeled goat anti‐rabbit IgG (Molecular Probes, Carlsbad, California, A11036) for TUJ1, and 594‐labeled goat anti‐rabbit IgG (Invitrogen, A11012) for NANOG and DAT, 488‐labeled goat anti‐mouse IgG (Invitrogen, A11001) for α‐synuclein. Nuclei were counterstained with DAPI (Dako, Hamburg, Germany) or NucSpot Live 650 Nuclear Stain (Biotium, Hayward, California). Immunofluorescence was visualized with an inverted fluorescence microscope IX73 (Olympus, Tokyo, Japan) or a confocal microscope Cell Voyager 7000 (Yokogawa, Tokyo, Japan). The fluorescence intensities were quantified using CV7000 analysis software.