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The brains were embedded into OCT compound (Sakura Fine Technical Co. Ltd., Tokyo, Japan) and frozen coronal sections (30 µm) were prepared.
Their brains were embedded into OCT medium (Leica Inc., Wetzlar, Germany) and quickly frozen in −80°C after resection.
Next, the brains were embedded into 2% agar in PBS and cut into 400-μm-thick coronal sections with a vibratome (Leica VT 1000 S).
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After washing (4×10 min) in Tris-SMB buffer, brains were embedded in agarose and cut into 60 µm sections.
The brains were embedded in paraffin and cut into 4 6 μm sections.
Brains were embedded in paraffin and cut into 5-μm sagittal sections.
For Nissl staining, paraformaldehyde-fixed brains were embedded in OCT Medium and cut into frozen sections (10 µM).
After the brains were embedded in paraffin, they were cut into 5-µm thick frontal sections.
Subsequently, the complete brains were embedded in paraffin and serially cut into coronal sections of 20 μm thickness.
The brains were embedded in 12% celloidin, cut into 40-μm serial sections using a sliding microtome and Nissl (1% cresyl violet) stained.
After post-fixation overnight at 4°C, the brains were embedded in agarose and then cut into 50 µm sections using a VT 1200S vibratome (Leica, France).
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