Immunofluorescence staining using SC samples

At 6- and 12-weeks post injury, euthanasia was performed using compressed CO₂ gas, which was injected at a rate of 30  70% of the chamber volume/min. Cardiac arrest was confirmed after euthanasia, and tissue sampling was performed. The injured SCs were obtained after transcardial perfusion with PBS and 4% PFA, fixed overnight in 4% PFA, and then incubated overnight in 15% and 30% sucrose solutions. Subsequently, the tissue samples were embedded in optimal cutting temperature (OCT) embedding matrix compound (Tissu-Tek; Sakura Finetek USA, Torrance, CA, USA) and snap-frozen in liquid nitrogen. Injured SC Sect. (4-µm thickness) were obtained and mounted on saline-coated slides. The SC sections were permeabilized with 0.1% Triton X-100 for 20 min at RT. Sections were blocked with normal goat or horse serum containing 0.1% Triton X-100 for 1 h at RT. Primary antibodies were diluted and incubated with a mixture of 0.1% Triton X-100 overnight at 4 ℃. After washing, Alex Fluor-488 or -594 and Cy5 (Life Technologies, Carlsbad, CA, USA)-conjugated secondary antibodies were diluted in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) at RT for 1 h. Slides were stained with DAPI, finally washed, and mounted using an antifade mounting reagent. Stained tissues were observed using a confocal microscope, LSM900 (Carl Zeiss, Oberkochen, Germany).