than the sections hybridized with sense probe were considered positively labeled for PAFR mRNA. For immunohistochemistry as a second staining after ISH, the sections were treated 3% hydrogen peroxide in PBS for 15 min, and Protein Block (Dako). Sections were incubated either with the anti-Iba1 rabbit polyclonal antibody (1:1000, Wako) or anti-GFAP rabbit polyclonal antibody (1:500, Dako) at 4uC for overnight. After washing with TBS, sections were incubated with biotin-conjugated goat anti-rabbit IgG (Dako) for 30 min at room temperature, followed by the addition of peroxidase conjugated streptavidin (Nichirei) for 5 min. Peroxidase activity was visualized by diaminobenzine. Statistical analysis All data are presented as means 6 SEM. The statistical significance of difference between values was determined by Student’s t test or analysis of variance (ANOVA) with appropriate post hoc tests. A p value less than 0.05 was considered to be statistically significant. 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PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10467