Distribution of ZIC proteins in normal tissues. Human adult-derived normal tissues were examined. (A, B, E, F) Meningeal membrane. (A, B, F) is comprised of neighboring sections. Image in (E) is a higher magnification of the boxed region in (A). Immunostaining with CXY2 (A, E), CXY2/vimentin (F), and hematoxylin and eosin staining (B) are shown. ZIC-like immunoreactivities are indicated as brown signals in (A, E, F). Vimentin-like signals are indicated by black in (F). Most Zic-like signals in cell nuclei are surrounded by the cytoplasmic Vimentin-like signals. a, arachnoid layer; d, dura mater. Scale bar, 100 μm. (C, D, G, H) Cerebellum. Immunostaining with CXY2 (C), ZC26 (G), and anti-ZIC4 (H), and hematoxylin and eosin staining (D) are shown. ZIC-like immunoreactivities are indicated as brown signals in (C, G, H). The majority of the cell nuclei in the granule cell layer are commonly stained with the three anti-Zic antibodies. In (H), the arrowheads indicate the cytoplasmic staining of the cells in Purkinje cell layer. The derivation of the cytoplasmic signals by the anti-ZIC4 antibody staining is unknown. gl, granule cell layer of cerebellum; ml, molecular layer of cerebellum; pl, Purkinje cell layer. Scale bar, 100 μm.