Immunohistochemical Staining Analysis Protocol

Immunohistochemical staining analysis was performed as previously reported [5 (link), 14 (link)]. Briefly, the samples were fixed with 3.7% formaldehyde (FUJIFILM Wako Pure Chemical Industries) and permeabilized with PBS containing 0.2% Tween-20 (FUJIFILM Wako Pure Chemical Industries). Samples were then blocked with PBS containing 4% Block Ace (DS Pharma Biomedical) and 0.2% Tween-20 and then incubated with primary antibodies (mouse anti-NGFR (1:200; Advanced Targeting System) and rabbit anti-SOX10 (1:500; Abcam)) overnight at 4°C. Samples were then washed with PBS containing 0.2% Tween-20 thrice and then incubated with secondary antibodies (anti-mouse Alexa Fluor-488 and anti-rabbit Alexa Fluor-555; 1:1000; Thermo Fisher Scientific). For nuclear staining, 0.2 μg/ml Hoechst 33342 (Dojindo Molecular Technologies) was added to the samples. The samples were placed on the stage of the inverted microscope (IX81; Olympus), and fluorescence was observed with an electron multiplying charge-coupled device camera (iXon; Andor). The observed images were analyzed using ImageJ software (National Institutes of Health; available at http://imagej.nih.gov/ij/).