Fig. 2. Localization of CXCR4 and SDF-1 mRNA and protein in postnatal cerebellar cortex and distribution of CXCR4 on granule cells in vitro. (A) CXCR4 mRNA expression was detected in both the EGL and IGL of P4 and P8 (not shown) cerebella. (B) SDF-1 mRNA expression was primarily limited to the pia mater overlying the cerebella of P4 and P8 (not shown) animals. A small amount of staining is also present throughout the cerebellum. (C) Control sections hybridized with sense RNA probes did not demonstrate expression of CXCR4 or SDF-1 (not shown). Bar represents 50 µm. (D) Immunostaining for CXCR4 using Cy3-conjugated secondary antibodies (red) demonstrates expression in the EGL and IGL of P4 and P8 (not shown) cerebella, consistent with localization of CXCR4 mRNA. Nuclei are localized with DAPI (blue). (E) Immunostaining for SDF-1 using Cy3-conjugated secondary antibodies (red) demonstrates high levels of expression in cells composing the pia mater of P4 and P8 (not shown) cerebella, consistent with localization of SDF-1 mRNA. There is also some staining of cells throughout the cerebellum, as observed for SDF-1 mRNA expression. The location of this staining suggests it may be in glial elements. Nuclei are localized with DAPI (blue). Bar represents 50 µm. (F) Confocal imaging of CXCR4-positive neurons detected with FITC-conjugated secondary antibodies indicates that granule cells express CXCR4 in vitro with punctate staining evident over the cell bodies and along the axonal process. (G) Cultured neurons stained with non-immune serum and detected with FITC-conjugated (green) secondary antibodies demonstrate no background (green) staining of cell bodies or processes; only nuclear staining with topro-3 is detected (blue). Bar represents 10 µm.