Immunofluorescence Labeling of Neurofilaments

Immunofluorescence for neurofilament was performed according to the previously described method (Jung et al., 2016 (link)). The sections were washed 3 times with PBS, and then exposed to 0.1% bovine serum albumin solution containing 0.1% Tween 20 in PBS for 4 hr at room temperature. Next, the sections were incubated overnight with a solution containing primary antibodies as antineurofilament rabbit polyclonal antibody (1:400, Sigma Chemical Co., St. Louis, MO, USA) for axons, After washing, the sections were incubated 2 hr with secondary antibodies as rhodamine-goat anti-rabbit secondary antibodies (1:800; Molecular, Probes, Eugene, CA, USA) for neurofilament. The sections were mounted and examined by a fluorescence microscope (Nikon Eclipse 50i, Nikon Inc., Melville, OR, USA).