Dmi400

Twenty-two hours after treatment, five rats from each group were anaesthetized and perfused with 200 ml normal saline and 200 ml 4% paraformaldehyde. Then, the brain was collected and post-fixed in 4% formaldehyde solution for 2 h, dehydrated, clarified, embedded in paraffin, and cut into sections (5 μm in thickness) to adhere to the slide (Leica 2035, Germany). The sections were routinely deparaffinized and stained with hematoxylin eosin (HE). The histological examination was performed under a light microscope; the nuclei in the sections appeared blue, while the cytoplasm appeared red. Five non-overlapping views of the cortex in each section were randomly observed under a microscope (Leica DMI400, Wetzlar, Germany) at 400-fold magnification for cell counting, and the results are presented as the means ± SD. The denatured cell index (the number of denatured cells/total cells) indicates the degree of damage.

Rabbit anti-rat pERK1/2 monoclonal antibody (1:100, # 4370) was purchased from Cell Signaling Tech. Co. Ltd., USA. Rabbit anti-rat ERK1/2 polyclonal antibody (1:100, BS-2637R) was provided by Bioss Biotech. Co. Ltd., Beijing, China. 3,3’-diaminobenzidine (DAB) and Power Vision two-step histo-staining reagent (PV-6001) were obtained from ZSGB-Bio. Co. Ltd., Beijing, China. Paraffin sections treated as above were de-waxed and washed according to routine procedures. The immunohistochemical procedures were performed in strict accordance with the manufacturer’s instructions. Under a microscope, the positive cells appeared with brown granules in the cytoplasm. Those sections to which 0.01 mmol/L PBS was added instead of primary antibody exhibited no positive responses. The positive cells were enumerated and averaged in five random views of cortex from four serial slices under a microscope at 400X magnification (Leica DMI400, Germany) The positive cell index (PCI = positive cells / total cells) was calculated and used to indicate the pERK1/2 and ERK expression levels.

At 24 h after treatment, 5 rats were randomly selected from each group and anesthetized with 10% chloral hydrate and then perfused with 200 ml of 4% formaldehyde via the heart. The brains were removed and fixed in 4% formaldehyde for 2 h, soaked in distilled water for 4 h, dehydrated using a graded ethanol series, hyalinized via dimethylbenzene, embedded in paraffin, and sectioned at a thickness of 5 μm. The sections were adhered to glass slides treated with poly-L-Lysine, which were stored at 4°C. After routine de-waxing and washing, the histological sections were stained with hematoxylin for 5 min, the color was separated with 1% hydrochloric acid alcohol for 20 s, the section were back to blue with 1% ammonia for 30 s, dyed with eosin for 5 min, dehydrated in increasing concentrations of alcohol, hyalinized with dimethylbenzene, and sealed with neutral gum. Under a microscope at 400X magnification (Leica DMI400, Germany), 5 non-overlapping views of the cortex in each section were randomly chosen, and the cells in these sections were counted and presented as mean ± SD. The denatured cell index (DCI = denatured cell number/total cell number) was used to indicate the degree of damage.