Retinectomy specimens from the 16 patients were removed from the globe after excision and immediately fixed in 4% paraformaldehyde in sodium cacodylate buffer (0.1 M; [pH 7.4]; Electron Microscopy Sciences, Fort Washington, PA) for a minimum of 24 hours. Control tissue was used from a 79-year-old female donor. Peripheral retina from an anatomic location similar to the retinectomy specimens was used for analysis. After they were rinsed in PBS, the specimens were orientated in 5% agarose (Sigma-Aldrich, St. Louis, MO) in PBS. Sections (100-μm-thick) were cut with a vibratome (Technical Products International, Polysciences, Warrington, PA) and incubated in normal donkey serum (1:20; Jackson ImmunoResearch, West Grove, PA) in PBS containing 0.5% bovine serum albumin (BSA; Fisher Scientific, Pittsburgh, PA), 0.1% Triton X-100 (Roche Molecular Biochemicals, Indianapolis, IN), and 0.1% sodium azide (Sigma-Aldrich) overnight at 4°C on a rotator (PBS, BSA, Triton X-100, and azide is referred to as PBTA). After the removal of blocking serum, primary antibodies were added in four sets of pairs: anti-glial fibrillary acidic protein (GFAP; 1:500; Dako, Carpinteria, CA) with anti-rod opsin (1:50; gift from Robert Molday, University of British Columbia, Vancouver, BC, Canada); anti-vimentin (1:500; Dako) with anti-medium to long-wavelength (M/L) cone opsin (1:2000; gift from Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD); anti-calbindin D (1:1000; Sigma) with anti-short-wavelength (S) cone opsin (1:2000; gift from Jeremy Nathans); and anti-cytochrome oxidase (CO; 1 μg/mL; Molecular Probes, Eugene, OR) with anti-synaptophysin (1:100; Dako). Additional antibodies were used to detect CD68 (1:200; Dako), neurofilaments (1:500; Biomeda Corp., Foster City, CA); protein kinase C (PKC; 1/100: Biomol Research Laboratories Inc., Plymouth Meeting, PA); growth-associated protein (GAP)-43 (1/200: Chemicon International, Temecula, CA) and cones, regardless of spectral sensitivity (7G6 culture supernatant, mouse monoclonal antibody used 1/100; gift from Peter MacLeish, Morehouse School of Medicine, Atlanta, GA).
After overnight incubation at 4°C on a rotator, sections were rinsed in PBTA and incubated again overnight at 4°C with the secondary antibody. Donkey anti-mouse and donkey anti-rabbit secondary antibodies were used for each combination of primary antibodies, conjugated to Cy2 or Cy3 (Jackson ImmunoResearch). All secondary antibodies were used at a dilution of 1:200, and all antibodies were diluted in PBTA. Selected sections were counterstained with DAPI (1:5000; Dako) for 5 minutes in one of the final washes. The sections were then rinsed, mounted in N-propyl gallate in glycerol and viewed on a laser scanning confocal microscope (model 1024; Bio-Rad, Hercules CA, or LSM 510, Carl Zeiss Meditec, Oberkochen, Germany).
The proportion of rod photoreceptors with obvious outer and inner segment material, and hence most capable of regeneration after reattachment, was calculated by averaging cell counts from two independent regions of each specimen.