Indirect immunocytochemistry using secondary antibodies conjugated to either cyanine, fluorescein, or 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) was performed on fixed cells or on ocular slices (4% paraformaldehyde) 5 to 15 days after plating, as previously described.
23 Primary antibodies included mouse monoclonal antibodies against β-tubulin isotype III (final concentration, 1:1000; Sigma), neurofilaments (1:100; Sigma), NeuN (1:500; Chemicon, Temecula, CA); S100β (1:200 Chemicon), vimentin (clone V9,; 1:200; Dako, Zug, Switzerland), P0 (Juan José Archelos, Universitätsklinikum, Graz, Austria), fibronectin (1:400; Sigma), smooth muscle antigen (1:400; Sigma), CD34 (1:200; Dako, Glostrup, Denmark), and rabbit antiserum glial fibrillary acidic protein (GFAP; 1:400; Genosys, Cambridgeshire, UK); and the mouse IgM monoclonal antibody against O4 (1:20; Roche Molecular Biochemicals, Mannheim, Germany). The rabbit polyclonal antibody against human nestin was a generous gift from Ron D. G. McKay (National Institutes of Health, Bethesda, MD),
24 and polyclonal antibodies against RPE65 and RGR were kindly provided by Andreas Wenzel (University of Zurich, Zurich, Switzerland). The secondary antibodies (Jackson ImmunoResearch, West Grove, PA) included fluorescein or cyanine-conjugated affinity-purified goat antibody to mouse IgG (1:100, 1:200, or 1:500) and AMCA-conjugated affinity-purified goat antibody to mouse IgM (1:100).