The handling of these birds was in agreement with the Australian code of practice for the care and use of animals for scientific purposes. All parts containing portions of the ION were imaged to ascertain the extent to which the IO nerves had taken on the tracer. To organize smooth mounts, the eyecup was washed with PBS and angiogenesis assay cut in two with one dorsalventral cut just temporal for the pecten. The pecten was vigilantly excised, to make sure minimum loss in retina. The retina was removed from the pigment and sclera epithelium before being processed using standard immunohistochemistry techniques. The retinas useful for the level bracket reports, in which we mapped the distribution of rEF terminals, came only from animals in which all of the IO nerves had taken up tracer, as verified by examining parts drawn from the entire scope of the ION. To get ready radial sections of the retina, the eyecup was rinsed with PBS and two 5 mm 5 mm sections, akin to the areas of the retina where efferent fibers are observed in greatest density, were cut out from either side of the pecten. These retinal pieces were separated from your sclera and put into 30 % sucrose solution at 4 C until equilibrated. The next morning they were kept at 20 C until sectioning, frozen on dry ice, and embedded in OCT medium. Chapters of 12 20 um Organism were cut on a cryostat, mounted on gelatin coated slides, and stored at 20 C. To recognize TCs we used the exact same anti parvalbumin antibody used by Fischer and Stell for this function. For smooth brackets marked with antiparvalbumin and both Fluoro Ruby, a blocking solution containing one of the BSA, 10 % normal goat serum, and 0. 1000 Tween 20 in PBS was requested 1 hr at room temperature. Retinas were incubated in mouse anti parvalbumin diluted 1:1000 in blocking solution for 7 days at 4 C, washed 3x in PBS, then incubated for 1 5 days at 4 C in secondary antibodies diluted 1:500 in PBS. Finally, the brackets were washed with PBS and coverslipped applying VectaShield hard set mounting medium. These day the slides were sealed using DPX. Retinal sections were processed for immunohistochemistry using techniques similar to those described for flat supports, except that sections were quenched with LY2484595 one of the glycine in 0. Three times Triton PBS for 15 min just before blocking option, and the incubation times for primary and secondary antibodies were paid down to overnight and 1 2 hrs, respectively. Flat brackets and stained sections were examined using an inverted confocal microscope hiring krypton and argon lasers. Images were converted to 24-bit TIF photographs in Fluoview software then utilized in Adobe Photoshop for change of brightness, contrast and sharpness. Images of antibody labeled muscle were often processed identically to pictures of the corresponding secondary only control. Since they were uniformly black, these are not shown.