Upregulation of CXCR2 ligands in the brain and spinal cord during EAE. a, Quantification of the mRNAs encoding each of the CXCR2 ligands by quantitative RT-PCR using RNA samples prepared from the brains or spinal cords of mice killed at the indicated time intervals after EAE induction (as shown in Additional file 1: Figure S1, the first clinical symptoms were observed on day 8). The means were compared with the Kruskal-Wallis test (P-values as indicated) followed by post hoc Wilcoxon tests. *Significantly different from day 3. Δ = fold change relative to day 3. n = 6 (days 3, 6, and 10), 5 (day 8), or 3 (day 12). b, Dark-field and bright-field micrographs showing in situ hybridization signals for CXCL1, CXCL2, or CXCL5 mRNA in the leptomeninges of EAE mice killed 6 days after EAE induction. Arrows show clusters of emulsion grains indicating the presence of cells expressing the transcripts. Scale bar = 50 μm. c, Double labeling for CXCL1 mRNA (black grains, in situ hybridization) and different cell type-specific markers (red-brown, immunohistochemistry) in brain sections from EAE mice. These images were taken at the level of the leptomeninges. Arrows indicate double-labeled cells. Arrowheads show examples of immunostained cell bodies negative for CXCL1 mRNA. Scale bar = 10 μm.