Statistical operations used the Vassar Statistics web server

essment, the peak area values obtained from the NMR chromatogram of each fatty acid were normalized using that of C19:0 tuberculostearic acid as an internal standard. Next, the amounts of each fatty acid in the hypothalamus extract, with and without CFA treatment, were calculated, subtracting the results of each negative control sample from those of the corresponding hypothalamus tissue extract. HPLC separation was performed on a Mightysil RP-18 GP column. The mobile phases were gradients of 10 mM ammonium acetate/methanol. The flow rate was set to 0.3 mL/min. Western blot analyses Western blotting was done as previously described with some modifications. Hypothalamus tissue was homogenized in homogenization buffer. Protein samples were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. GPR40 was then assessed using rabbit polyclonal primary antibodies, and glial fibrillary acidic protein was detected using mouse monoclonal primary antibodies. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control and was detected using primary antibodies. Blots for GPR40 and GFAP were incubated overnight with the primary antibody at 4uC in Tris-buffered EMA401 saline containing 0.1% Tween-20 and blocking agent. After washing, blots were incubated with horseradish peroxidase -conjugated anti-rabbit IgG for GPR40 and HRP-conjugated anti-mouse IgG for GFAP and GAPDH for 1 h at room temperature. Immunoreactive bands were visualized using a Light-Capture system with an ECLTM Western Blotting Analysis System. The signal intensities of immunoreactive bands were analyzed using a CsAnalyzer. GPR40, which was prelabeled with the Zenon Alexa Fluor 594 Rabbit IgG Labeling Kit. NeuN, GFAP, 18347191 proopiomelanocortin , c-Fos and bendorphin were stored overnight at 4uC, at which time the antibody was 23127512 diluted in reaction buffer. The next day, sections were washed with PBST and incubated in secondary antibody conjugated with AlexaFluor 488 and/or 594 at room temperature for 2 h, at which time the secondary antibody was diluted with reaction buffer. Finally, sections were washed with PBST and coverslipped with Perma Fluor, and immunoreactivity was detected with a confocal fluorescence microscope. In the immunohistochemical control studies, no staining was detected when the corresponding primary or secondary antibody was omitted. Statistical analyses Data were expressed as mean 6 S.E.M. Significance differences were evaluated by one-way analysis of variance followed by Dunnett’s or Scheffe’s multiple-comparison tests for comparisons between more than three groups or by Student’s t-test for comparison between two groups. A p value of,0.05 was regarded as significant. Results Development of hyperplasia, mechanical allodynia and thermal hyperalgesia after CFA injection Long-lasting paw hyperplasia, persistent mechanical allodynia and thermal hyperalgesia were elicited in CFAtreated mice, compared with saline-injected control mice, appearing on day 1 and continuing until day 14. No pain behavior was observed in saline-injected mice. Brain tissue preparations Mice were deeply anesthetized with sodium pentobarbital and perfused transcardially with phosphate-buffered saline, pH 7.4, followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Brain sections were collected, post-fixed in 4% paraformaldehyde for 3 h, and dehydrated in 10% sucrose at 4uC for 3 h, and 20% sucrose at 4uC overnight. The following day, t