Rat anti mbp monoclonal antibody

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 remaining rats in each group were perfused with 4% chloral hydrate (1 ml/100 g, P57). Hippocampi were placed in a precooled labeled EP tube and then quickly stored at −80°C. Four rat tissues were taken from each group for protein extraction, and the remaining tissues were frozen for other purposes. Detailed steps of the Western blotting method have been previously described (Ni et al., 2018 (link)).
Briefly, polyvinylidene fluoride membrane blots were incubated with one of the following antibodies: goat anti-ZnT3 (1:1000, Santa Cruz), rabbit anti-GPR39 polyclonal antibody (1:1000, Biorbyt), rat anti- MBP monoclonal antibody (1:2000, Abcam), mouse anti-GAPDH monoclonal antibody (1:5000, Proteintech) or rabbit anti-β-tubulin polyclonal antibody (1:5000, Proteintech) in TBST contain 5% non-fat dry milk overnight at 4°C. Membranes were then incubated with secondary antibodies for approximately 2 h. ECL chemiluminescence kit A and B were mixed in equal volume, and PVDF membranes were immersed in the luminescent liquid for approximately 2 min, and then the film was placed in an automatic developing device for exposure (LAS 4010, GE Healthcare Life Sciences, Little Chalfont, United Kingdom). Grayscale values of each band were analyzed using ImageJ image processing software.