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The brain tissue sections were firstly fixed by methanol for 5 min, then stained with the Cresyl Echt Violet staining kit (American MasterTech, catalog # AHC0443) and incubated for 5 min; next, the slides were rinsed in 2 changes of distilled water (15 s each) to remove the excess stain; then the slides were rinsed using 100% ethanol (10 s). At last, the slides were coverslipped with mounting medium.
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