The brains were fixed in 4% paraformaldehyde (Nacalai tesque, Kyoto, Japan) in 0.2 M PBS (pH 7.4) and then embedded in paraffin (Merck Ltd., Frankfurt, Germany) using standard procedures. The paraffin blocks were cut into 5-μm sections. Sections were incubated overnight at room temperature with rabbit anti-GFAP polyclonal antibody (1:300; Abcam, Cambridge, UK), rat anti-MBP monoclonal antibody (1:50; Abcam, Cambridge, UK), or mouse anti-synaptophysin monoclonal antibody (1:100; Millipore, Darmstadt, Germany), followed by an appropriate secondary antibody for 2 h at room temperature.
Fluorescent images were acquired using an FV1000-D IX81 confocal laser microscope (Olympus, Tokyo, Japan). The average number of GFAP positive (GFAP+) cells in the corpus callosum at the striatal level on P15 or P30 was manually counted in five randomly chosen fields (120 μm × 260 μm) in each of three sections per animal, spaced 350 μm apart28 (link). The ratios of the areas positively stained with MBP in the cingulum and synaptophysin in the dorsal hippocampus on P15 or P30 were analyzed using Image J software (National Institutes of Health, US) in three sections per animal, spaced 350 μm apart29 –33 (link).
Fluorescent images were acquired using an FV1000-D IX81 confocal laser microscope (Olympus, Tokyo, Japan). The average number of GFAP positive (GFAP+) cells in the corpus callosum at the striatal level on P15 or P30 was manually counted in five randomly chosen fields (120 μm × 260 μm) in each of three sections per animal, spaced 350 μm apart28 (link). The ratios of the areas positively stained with MBP in the cingulum and synaptophysin in the dorsal hippocampus on P15 or P30 were analyzed using Image J software (National Institutes of Health, US) in three sections per animal, spaced 350 μm apart29 –33 (link).