Fluoro mounting medium

At 3 or 12 weeks after ONC, tissues were collected after perfusion for immunostaining. Frozen sections (25 μm thickness) were prepared using a cryostat microtome (Leica, Deer Park, IL, USA ) and fixed in 4% PBS-buffered paraformaldhyde solution, and permeabilized using 0.3% Triton-X 100 and 3% goat serum in PBS for immunostaining. Tissue samples were incubated with primary antibodies diluted in PBS that contained 3% goat serum at 4 °C overnight. The primary antibodies used were rabbit anti-GAP43 (1:500, Cell Signaling, Denve, MA, USA), anti-β-III tubulin (1:500, Cell signaling) and anti-MBP (1:500, Cell signaling). After washing 3 times with PBS, the samples were incubated with Alexa Fluor 488 (1:200, green) or Alexa Fluor 555 secondary antibody (1:200, red) (Molecular Probes, Waltham, MA, USA) for 1 h at room temperature. The samples were mounted in fluoro mounting medium (Millipore, Burlington, MA, USA) and images were taken with a Zeiss LSM700 confocal microscope (Kuehnstrasse, Hamburg, Germany). The fluorescence images were analyzed using ImageJ from the National Institute of Health (Bethesda, MD, USA) to analyze the fluorescence intensities of the tissues compared with that of the control after subtracting the background value.

At 4 weeks after ONC, tissues were collected after perfusion for immunostaining. Frozen sections (25 μm thickness) were prepared using a cryostat microtome (Leica) and fixed in 4% PBS-buffered polyformaldehyde solution, and permeabilized using 0.3% Triton-X 100 and 3% goat serum in PBS for immunostaining for GAP-43, a regenerating axon marker. Tissue samples were incubated with anti-GAP43 antibody (1:500, Cell Signaling) diluted in PBS containing 3% goat serum at 4 °C overnight. After washing 3 times with PBS, samples were incubated with Alexa Fluor 488 goat anti-rabbit (1:200) secondary antibody (Molecular Probes, Thermo Fisher Scientific Inc.) for 1 h at room temperature. Samples were mounted with fluoro mounting medium (Millipore, Carlsbad, CA, USA) and images were taken with a Zeiss LSM700 confocal microscope (Oberkochen, Germany).