(A) Western blot of protein homogenates from HEK293 cells transfected with a mouse Shh (HEK-Shh) or control vector (HEK-mock) and from hypothalamic (Hpt), cerebellar (Cb) or cerebrocortical (Ctx) tissues of an adult mouse with the C9C5 antibody, and was reproduced with tissues from three other animals. A 22 kDa band, the expected size for the aminoterminal fragment of Shh, was detected in HEK-Shh cells and in tissues but was absent in HEK-mock cells and in blocking experiments (n = 3). A 47 kDa signal corresponding to the uncleaved Shh protein was detected in HEK-Shh cells but not in tissues. Actin served as a loading control. (B-C) Low-magnification photomontages of coronal brain sections of adult mice showing strong C9C5 immunoreactivity in almost all brain regions (B), which was absent in blocking experiments (n = 3) (C). Staining was replicated in the twenty adult mice used in this study. Nuclei stained with DAPI. 3V, third ventricle; Hpt, Hypothalamus; Ctx, cerebral cortex; Cb, cerebellum; cc, corpus callosum; DG, dentate gyrus of the hippocampus.