Figure 2 Astrocyte activation profile is modified in Sema4B−/− mutant mice. A, Representative examples of a horizontal section of wild-type (n = 7) and Sema4B mutant cortices were stained with GFAP (green) and Hoechst stain (blue) 7 d after injury. Note the reduced activation indicated by GFAP staining near the site of injury in the mutant mouse. Scale bar, 100 μm. B, a representative coronal section of Sema4B+/− with or without injury stained with X-gal histochemistry. Scale bar, 200 μm. The gray boxes mark the area of counting that was used in all sections. C, Quantitation of X-gal-positive cells/area in the noninjured cortex of Sema4B+/− and Sema4B−/− mice. D, Quantitation of the number of X-gal-positive cells per area in the injured cortex of Sema4B+/− and Sema4B−/− mice. E, Representative example of cortex after injury in low magnification, stained with anti-S100β. The black boxes mark the area of counting (in F and G). Scale bar, 200 μm. Representative example of Sema4B+/− and Sema4B−/− mice near the site of injury stained with S100β is also shown. Scale bar, 20 μm. Note that the typical astrocyte hypertrophy is detected in both Sema4B+/− and Sema4B−/−. F, Quantitation of the number of S100β-positive cells per area in the noninjured cortex of Sema4B+/− and Sema4B−/− mice. G, Quantitation of the number of S100β-positive cells per area in the injured cortex of Sema4B+/− and Sema4B−/− mice. H−J, Relative mRNA levels were measured using real-time PCR analysis (mean ± SEM) of cortical tissue at the site of injury 1 d postinjury (I: LCN2, Sema4B+/−, n = 4; Sema4B−/−, n = 5) and 7 d postinjury (J: GFAP; H, vimentin; n = 5; Sema4B+/−, n = 5; Sema4B−/−, n = 6). GFAP LCN-2 and vimentin in each sample were normalized relative to GAPDH (similar results were obtained when samples were normalized to ALDH1L1). Note the change in postinjury astrocyte activation profile in the Sema4B−/− cortex (low GFAP, normal activation of vimentin, and higher activation than normal for LCN2. For GFAP, p = 0.0081; for LCN2, p = 0.0357.