0.1 m phosphate buffer

The primary cultured cells were fixed for 1 min with 4% paraformaldehyde in 0.1 M phosphate buffer (Wako, Osaka, Japan) at room temperature. After several washes, they were solubulized in PBS with 0.1% Triton X-100 and incubated overnight at room temperature with the following primary antibodies: mouse monoclonal anti-Neuronal ClassIII β-Tubulin (Tuj1) antibody (1:500; Covance, Princeton, NJ, USA), mouse monoclonal anti-CD11b antibody (1:500; BD bioscience, Franklin Lakes, NJ, USA), mouse monoclonal anti-GFAP antibody (1:200; Sigma-Aldrich, St. Louis, MO, USA), and rabbit polyclonal anti-Nogo receptor (NgR1) antibody (1:200; abm, Richmond, BC, Canada). After three rinses with PBS, the cells were incubated for 60 min at room temperature with the following secondary antibodies: Alexa Fluor 594-conjugated goat anti-rabbit (Invitrogen), and Alexa Fluor 488-conjugated donkey anti-mouse (Invitrogen). The cells were rinsed in PBS, mounted with Fluorsave (Calbiochem, San Diego, CA, USA), and observed under a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).

Cells were seeded onto a 60-mm dish at 1×10⁶ cells/dish and treated with various reagents for 48 h. Then, the cells were fixed for 1 h at 4°C with 2.5% glutaraldehyde (TAAB Laboratories Equipment, Ltd.) in 0.1 M phosphate buffer (pH 7.3) (prepared with monobasic sodium phosphate and dibasic sodium phosphate; Wako Pure Chemical Industries), and fixed subsequently for 1 h at RT in 1% osmium tetroxide (Nisshin EM Co., Ltd.), dehydrated in graded ethanol (30-100%), and embedded in Quetol 812 epoxy resin (Nisshin EM Co., Ltd.) at 60°C for 2-4 days. ultrathin sections (60 nm) were obtained using an Ultracut J micro-tome (Reichert Jung), and the sections were stained with 4% lead nitrate (RT, 5 min) and saturated uranium acetate (RT, 10 min) and imaged using a transmission electron micro-scope JEM-1200EX II (JEOL, Ltd.) (magnification ranging from ×1,000 to ×10,000). All images were captured on films (Electron-microscopic film FG; Fujifilm).