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2��C. Because of individual variations, the animals used in this study were chosen with the HPL response time shorter than 20 sec but longer than 6 sec under the normal condition. Rats were deeply anesthetized with chloral hydrate (300 mg/kg, ip), transcardially perfused with 0.1 M phosphate-buffered saline (PBS) followed by cold 4% paraformaldehyde in PBS. The lumbar-sacral enlargement of the spinal cord and the brain were postfixed in 4% paraformaldehyde and dehydrated in 20% sucrose solution overnight. Sections at 30 ��m thickness were prepared for c-Fos IHC staining. Sections were incubated with 0.3% H2O2 in PBS (0.5 hr), blocked in 5% normal goat serum (60 min), and incubated overnight with primary antibody c-Fos (1:100, Santa Cruz), then with biotinylated goat anti-rabbit IgG. Finally, sections https://en.wikipedia.org/wiki/Evodiamine were developed with DAB (Sigma, USA) staining. c-Fos positive neurons in the spinal dorsal horn, the S1 area of the cortex, and the amygdala were counted in five representative sections. Five areas (126.50 ��m2 per area) were chosen for cell counts from each section. Rats were anesthetized with chloral hydrate (250 mg/kg, ip) and decapitated at each time point. The tissues from the bilateral spinal cord, the SI area of the cortex, and the amygdala were immediately removed. The sectioned http://www.selleckchem.com/products/Dasatinib.html tissues were homogenized at 4��C in homogenizing buffer and sonicated to dissolve the tissue completely as previously reported (Jiang et al., 2009). Then, the amounts of protein were determined by BCA kit (Pierce, USA). The levels of ERK1/2 phosphorylation (p-ERK1/2) and protein expression were determined using WB as described previously (Zhang et al.,2007). Samples containing an equal amount of total protein were loaded on 10% SDS-PAGE gel. After transferring to nitrocellulose membranes (Schleicher & Schuell, USA), unspecific binding was blocked by incubating in blocking buffer for 1 hr. The blots were then incubated with primary antibodies overnight at 4��C. The following antibodies at a 1:1,000 dilution were used: rabbit anti-mouse p-ERK1/2, rabbit anti-mouse total ERK1/2 (T-ERK1/2, Promega, USA), or mouse anti-��-actin (Sigma, USA). After incubating with an anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody, protein was visualized using ECL-plus Kit (PerkinElmer Life Science, USA). Data were presented as mean �� SD (standard http://www.selleckchem.com/screening/epigenetics-compound-library.html deviation). Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by individual post hoc multiple comparisons. A statistical difference was accepted as significant at P