Fig 1.
Immuno-labeling for eCry1b in the retina of European Robins.
Retina of a robin (a) in migratory state, and of one (b) after the end of the migratory period. In (a), there is eCry1b labeling in the ganglion cells (layer 6) and the few displaced ganglion cells (layer 4). eCry1b label is located in the cytosol of the cell; the nuclei, indicated by arrows, show no label. In (b), the labeling in the ganglion cells is very low (see also Fig B in S1 Supporting Information). (c-e) Retinal ganglion cell layer in a robin in migratory state, triple-labeled for (c) eCry1b, (d) NeuN, and (e) DAPI. Practically all cells in the ganglion cell layer express eCry1b. Layers of the retina: 1, photoreceptor outer and inner segments; 2, outer nuclear layer; 3, outer plexiform layer; 4, inner nuclear layer; 5, inner plexiform layer; 6, ganglion cell layer; 7, optic nerve fibre layer. The scale bar is 50 μm for all panels.
Fig 2.
Electron-microscopic image of a ganglion cell, and Western blots.
(a) Ganglion cell in the retina of a robin in migratory state. eCry1b labeling is visualized with diaminobenzidine and silver intensification, visible as dark dots (some marked by arrows). Other subcellular components cannot be identified; eCry1b is probably free in the cytosol and also bound to membranes. (b, c) Western blots of the robin retina, indicating eCry1b (b; ~65 kDa) and eCry1a (c; ~70 kDa) in the cytosolic and in the membrane fraction. Both cryptochromes were detected in the same blot; the part showing eCry1a was already published in [15]. F1, cytosolic fraction; F2, membrane fraction; F3; nuclear fraction; F4, cytoskeletal fraction; T, tongue tissue from the same bird as control. (d) Western blot of purified eCry1a and eCry1b that had been treated with the eCry1b antiserum as control for the specificity of the antiserum, indicating that there is no cross-reactivity of the eCry1b antiserum with eCry1a.
Table 1.
Robins used in this study.