Specimens were deparaffinized in xylene, rinsed with 0.1 mM phosphate-buffered saline (PBS; pH 7.4), incubated with 0.4% trypsin at 37°C for 20 minutes to provide better infiltration of antibodies according to the manufacturer’s recommendation, rinsed with PBS, and blocked with 2% bovine serum albumin-PBS at room temperature for 30 minutes. Specimens then were incubated sequentially with one of the primary antibodies: rabbit monoclonal anti-neurofilament 200 (NF200: Sigma Chemical Co., St. Louis, MO) diluted 1:250 or mouse monoclonal anti-human neurofilament (SMI31: Sternberger Monoclonals, Baltimore, MD) diluted 1:250. NF200 recognizes both phosphorylated and dephosphorylated forms of the 200-kDa neurofilaments, whereas SMI31 reacts only with a phosphorylated epitope in extensively phosphorylated forms of this polypeptide. After three washes in PBS, specimens were incubated with secondary antibodies, either Texas red–conjugated anti-rabbit IgG for NF200 or FITC-conjugated anti-mouse IgG for SMI31. Specimens then were washed and mounted. A confocal laser microscope (TSC4D; Leica Microsystems, Wetzlar, Germany) was used for observation.