Antigen retrieval was performed by heating (90°C), for 30 minutes, unstained sections immersed in citrate buffer (pH 6.3) and then preincubated with 2% normal horse serum, 0.1% bovine serum albumin (BSA), and 0.4% Triton X-100 in 0.01 M PBS for 1 hour. For immunodetection of glial cells, sections were incubated overnight at 4°C with a mouse monoclonal anti–vimentin antibody (1:100; Dako, Carpinteria, CA) or a mouse monoclonal anti–GFAP antibody conjugated to Cy3 (1:1200; Sigma Chemical Co., St. Louis, MO). To analyze plexiform layers, a mouse monoclonal anti–synaptophysin antibody (1:100; Dako) was used. Some sections were treated without primary antibodies to confirm specificity. An anti–mouse secondary antibody conjugated to Alexa Fluor 568 (1:500; Molecular Probes, Eugene, OR) was used to detect vimentin and synaptophysin. After immunostaining, nuclei were stained with the fluorescent dye Hoechst 33342 (5 μg/mL in 1% dimethyl sulfoxide) and were placed under a microscope (BX50; Olympus, Tokyo, Japan) for observations.