Fv1000 d ix81 confocal laser microscope

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 apart^{28 (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 apart^{29 –33 (link)}.

The Paraffin blocks were sectioned at a thickness of 5 μm and subjected to immunohistochemical analysis. For the evaluation of phosphorylated mTOR (p-mTOR), an anti- phospho-mTOR (Ser2448) antibody (1:100, #ab109268, Abcam, Cambridge, UK) was used as the primary antibody, and 4’,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Sections were incubated overnight at room temperature with the primary antibody, followed by an appropriate secondary antibody for 2 h at room temperature. Fluorescence images taken by FV1000-D IX81 confocal laser microscope (Olympus, Tokyo, Japan) were analyzed using Image J software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) to determine the percentage of p-mTOR-positive area in the labyrinth zone of the mouse placenta.