Dmi400

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.