Eyes were marked dorsally, fixed in 4% paraformaldehyde for 2 hours, placed in 70% ethanol overnight, and wax embedded. Orientation was so as to section ventrodorsally and reveal the nasotemporal axis; 1 of every 10 sections (6-μm sections) was collected.
Series were stained with hematoxylin and eosin, rhodopsin, isolectin IB4 (Griffonia simplicifolia), or glial fibrillary acidic protein (GFAP). Sections were permeabilized (1% with Triton X-100 plus cations in PBS, blocked with 0.3% H2O2) and washed in PBS. Primary antibodies were diluted in PBS Triton X/cations: rhodopsin Rho-4D2 (1:1000; the kind gift of David Hicks, INSERM, City University Hospital, Strasbourg, France), isolectin IB4 Griffonia simplicifolia (1:100; Vector Laboratories, Burlingame, CA), GFAP (1:1000; Dako, Botany, NSW Australia) for incubation overnight at 4°C. Slides were washed, incubated with streptavidin-horseradish peroxidase (50% in PBS, LSAB Kit; DakoCytomation) at room temperature for 1 hour, washed, incubated in 3,3′-diaminobenzidine (DAB) substrate, washed, dehydrated, cleared, and mounted.