Paraffin sections 7-μm thick of the ON were dewaxed and rehydrated. Following boiling citrate buffer (pH 6.0)-antigen retrieval, the sections were blocked with 2% donkey serum and incubated with mouse monoclonal antibody to neurofilaments (SMI312: Sternberg Monoclonals, Lutherville, MD, USA) for intact axons, rabbit anti-human glial fibrillary acidic protein (GFAP; ab7262; Abcam, Cambridge, MA, USA) for glial scarring at 1:1000 dilution, rabbit anti-myelin basic protein (MBP; NB100-872; Novus International, Brussels, Belgium) for myelination and rabbit antibody to the ionized calcium channel IBA-1 (Wako Chemicals, Osaka, Japan) for inflammatory cells at 1:500 dilution overnight at 4°C. Slides then were washed and incubated with the appropriate fluorescent-labelled secondary donkey antibody (Jackson Immunoresearch, West Point, PA, USA) for 4 hours at room temperature, followed by extensive washing. Slides were mounted with aqueous mounting medium and examined at the appropriate wavelengths using an Olympus four-channel confocal microscope (Center Valley, PA, USA).