Anti neurofilament 200

The primary antibodies used in this study were as follows: anti-NDE1 (Proteintech, 1:500); anti-Olig2 (Millipore, 1:100); anti-PDGFRα (Santa Cruz, 1:100); anti-CC1 (Millipore, 1:100); anti-CNPase (Sigma, 1:300); anti-MBP (Millipore, 1:300); anti-Neurofilament 200 (NF, 1:300) (Sigma); anti-GFP (Santa Cruz, 1:300); anti-copGFP (Evrogen, 1:500); anti-HA (Sigma, 1:500); anti-dynein, 74-kDa intermediate chains, cytoplasmic (Millipore, 1:500); and anti-GAPDH (Santa Cruz, 1:300). The secondary antibodies were as follows: horseradish peroxidase (HRP)-conjugated anti-immunoglobulin G (IgG) (Cell Signaling, 1:1000); and Alexa Fluor 488-conjugated anti-IgG, Alexa Fluor 568-conjugated anti-IgG, and Alexa Fluor 350-conjugated anti-IgG (Molecular Probes, 1:1000).

Terminally anesthetized rats and mice were perfused with PBS, followed by 4% paraformaldehyde with 0.025% picric acid in PBS (PFA solution) and L4/L5 DRGs rapidly dissected. Post-mortem human lumbar DRG from a non-diseased donor (ND03910) was obtained via the National Disease Research Interchange program. All DRGs were post-fixed in PFA solution overnight and subsequently transferred into 20% sucrose in PBS for 24 h. Twelve-fifteen micrometer frozen sections were prepared, blocked for 1 h, and then incubated overnight at 4 °C in 1% BSA with 0.1% Triton X-100 in PBS with the following primary antibody: anti-peripherin (rabbit, 1:400, Chemicon), anti-neurofilament-200 (mouse, 1:300, Sigma-Aldrich), anti-substance P (SP, goat, 1:500, Santa Cruz Biotechnology), and anti-CASP6 (rabbit, 1:500, Cell Signaling Technology or rabbit, 1:1000, Abcam, Cambridge, UK). Primary antibodies were followed by Cy3- or FITC-conjugated secondary antibodies (1:400; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) or FITC-conjugated IB4 (10 μg/ml; Sigma-Aldrich). NeuroTrace green fluorescent Nissl stain (Invitrogen, Carlsbad, CA) was used to reveal the morphology of the somata of neuronal cells. Sections were examined under a Zeiss or Nikon fluorescence microscope. The specificity of the CASP6 antibody was tested in our previous study^{23 (link)}.

The proliferation of SCs was evaluated by seeding the cells at a density of 8.0 × 10⁴ cells/well into 6-well plates, which were then cultured by DMEM supplemented with 1% penicillin/streptomycin and 10% FBS at 37 °C in a humidified atmosphere containing 5% CO₂. The morphology of SCs after photobiomodulation was depicted via immunofluorescence staining technology. Briefly, the samples were fixed with 4% paraformaldehyde for 24 h at 4 °C. The mouse anti-S100 (1:500, ABCAM, USA) and anti-neurofilament 200 (1:80; Sigma, USA) antibodies were applied as primary antibodies overnight at 4 °C. Secondary antibodies, including goat anti-rabbit IgG (FITC)/goat anti-mouse IgG (TRITC) (Dako Japan) and DAPI (1:200, Life Technologies), were applied for 1 h at room temperature. The images were acquired by using a laser confocal microscope (FV10i-oil, push around, Tokyo, Japan).
For real-time live-cell analysis of SCs, the cells were seeded into 96-well culture plates at a density of 3 × 10³ cells/ml. Cell viability was measured using an IncuCyte S3 (Essen BioScience MA, USA) at 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, and 60 h time points during the assay. By counting the total number of cells in each subregion and dividing it by the area of the subregion, an estimate of the local cell density in that subregion was obtained.