Nbp1 49461

Dorsal root ganglia (DRG) and spinal cords were harvested from injured mAtf3pro/RmGFP reporter mice perfused with ice-cold PBS followed by cold 4% paraformaldehyde (PFA). In addition, sciatic nerves were harvested distal to the crush in treated mAtf3pro/RmGFP reporter mice perfused with cold 4% PFA. Perfused tissues were post-fixed for 3 hr at 4°C, and cryoprotected with 30% sucrose in PBS overnight. DRG sections (10 μm), spinal cord sections (20 μm) and sciatic nerve sections (60 μm) collected, blocked and permeabilized with 1% Triton X-100 in blocking buffer (Roche Diagnostics) for 1 hr at RT. Sections were incubated with rabbit polyclonal antibody against ATF3 (Santa Cruz Biotech; sc-188; 1:1000), TRPV1 (Alomone; ACC-030; 1:1000), SCG10 (Novus; NBP1-49461; 1:2000), 53BP1 (Novus; NB100-304; 1:2000), chicken polyclonal antibody against NF200 (Millipore, AB5539; 1:2000), rat polyclonal antibody against Laminin-γ (Millipore; MAB-1914P; 1:1000), DyLignt_594 conjugated Isolectin B4 (VectorLab; DL-1207; 1: 200), or mouse monoclonal antibody against phospho-Histone H2A.X (Ser139)(γ-H2AX; gamma-H2AX) (Millipore; 05-636; 1:400) at 4°C overnight and then incubated with Alexa Fluor 568 goat antibody against rabbit IgG, chicken IgG or rat IgG for 1 hr at RT. Images were acquired using a Nikon Eclipse 80I Microscope.

Cell and tissue extracts were collected in radioimmunoprecipitation (RIPA) lysis buffer and protein concentrations were measured by Bradford assay (Bio-Rad) to equalize loading. Samples were boiled for 5 minutes in SDS buffer before being analyzed on polyacrylamide gels, transferred to nitrocellulose membrane (0.2 micron) and blocked for one hour in 5% milk 0.1% tween-20 tris buffered saline (TBST). All membranes were incubated overnight with primary antibody (GAPDH – Abcam # Ab8245; Stathmin-2 – Novus # NBP1-49461) immunoblots were then washed three times with TBST and probed with horseradish peroxidase conjugated secondary antibodies diluted 1:5,000–1:10,000 in 5% milk for 1 hour at room temperature before being exposed to films or imaged on a LICOR Fc imager.

Adult mice were anaesthetized and perfused with 4% PFA in PBS. For cross-section analyses, the sciatic nerve was harvested at 7 days after crush when degeneration of pre-existing axons of mature neurons was complete (Di Maio et al., 2011 (link); Shin et al., 2012 (link)). DRGs were dissected and post-fixed in fixative at 4 °C for 5 min and the sc iatic nerve was post-fixed overnight, and then cryoprotected in 30% sucrose (wt/vol) for 24 hr at 4°C. The nerve was sectioned into 10 μm thickness at every 1 mm from 1 mm proximal to injury site (−1) to 6 mm distal to injury site. The sectioned nerves were stained with anti-GFP antibody and the total number of axons was quantified at each distance by using ImageJ software. The section 1 mm proximal to injury site served as THE control and the axon number in other sections was normalized to the control for each animal to assess the regeneration rate.
For the longitudinal sections assay, sciatic nerves were dissected at SNL D3 and postfixed with 4% PFA. Longitudinal sections were stained with SCG10 (Novus Biologicals, NBP1–49461). SCG10 fluorescence intensity was measured by NIH ImageJ along the distance as previously described (Di Maio et al., 2011 (link); Shin et al., 2012 (link)). An SCG10 intensity plot was drawn with average intensities calculated from non-overlapping 10 μm regions and normalized to that observed at the crush site.