Fig. 5. Anti-Sema5A antibody characterization and Sema5A localization at the optic disc. (A) In immunoblots, the Sema5A antibody recognized ECD-Fc protein (lane 1), Sema5A-myc from transfected 293 cells (lane 2), and yielded a band of the expected size for Sema5A (135 kDa) from E14 retina (lane 3). (B) Anti-Sema5A staining of live Sema5A-myc-expressing 293 cells. Top row shows Sema5A-myc-expressing cells stained with Anti-Sema5A, and the bottom row shows Sema5A-myc cells similarly treated with pre-immune serum. (C) Bright-field image of the optic disc. Arrowheads point to retinal pigment epithelium cells (^* marks the center of the optic disc). (D) Same optic disc as in C. Sema5A immunoreactivity was localized to the peripheral rim of the optic disc. Image compiled from optical sections through a depth of 25 µm at the optic disc. Scale bars: (B) 25 µm; (C-D) 50 µm.