Fig. 8

Localization of Mpeg1 and microglia/macrophage. Representative images of double staining of in situ hybridization for Mpeg1 and immunohistochemistry for Iba1 using the cervical cord at 3 dpi. Whole image of the spinal cord section (a, e, i, m) or magnified images of the insets (b-d, f-h, j-l, n-p) of sham (a-d, i-l) or pyramidotomy (e-h, m-p) groups, which were injured at P7 (a-h) or 8 weeks of age (i-p), are shown. Bright field (a, b, e, f, i, j, m, n), red channel (Iba1 signal, c, g, k, o), or merged images (d, h, l, p) are shown. Blue/purple indicates signal for Mpeg1. Right side of the spinal cord is marked with black pigment. Yellow arrowheads indicate colocalization of Mpeg1 and Iba1, and blue arrowheads indicate Mpeg1 signal which do not colocalize with Iba1 signal. Scale bars: 200 μm (a, e, i, m) or 50 μm (b-d, f-h, j-l, n-p). Dorsal is at the top, ventral is bottom, left is to the left, and right is to the right. Representative images of negative controls using sense probe are shown in Additional file 3. Note that whole area of the spinal cord is weakly stained with blue/purple in neonatal samples in sense probe, suggesting that the weak blue/purple signal in neonatal samples is nonspecific background. Because floating slices shrank during hybridization, the edge of the slices were folded in most cases when they were mounted on glass slides