Eyes were removed from control and glaucomatous dogs: two glaucomatous eyes were removed from our colony Basset Hounds, and two additional glaucomatous eyes were removed from clinical Basset Hound patients with primary closed-angle glaucoma not related to our colony dogs. Eyes were immersion fixed in 4% paraformaldehyde in 0.1 M PO4 buffer for 1 day and then embedded in paraffin and sectioned at 7 μm. For antibody staining, retinal tissue sections were deparaffinized, washed in phosphate-buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2 HPO4, 1.47 mM KH2PO4), and incubated in blocking solution containing 5% goat serum, 0.4% bovine serum albumin (BSA; Sigma, St. Louis, MO), and 0.2% Triton X-100 (Fisher Scientific, Houston, TX) in PBS. Tissue was incubated overnight at 4°C with an anti-glial fibrillary acidic protein (GFAP, 1:250 dilution; reactive glial cell marker; Dako, Carpinteria, CA) primary antibody diluted in blocking solution. The preparations were then rinsed with PBS and incubated with a secondary antibody (1:1000 dilution; Jackson ImmunoResearch, West Grove, PA) in blocking solution for 90 minutes at room temperature. Sections were washed in PBS and coverslipped (Gelmount mounting media; (Fisher Scientific). Negative controls were carried out in parallel by omission of the primary or secondary antibody. Nonspecific labeling was not observed under these conditions.
Optic nerves were harvested after eye removal and were dissected. Three-millimeter-thick segments obtained 1 mm posterior to the globe were rinsed in cacodylate buffer and postfixed in 2% osmium tetroxide in cacodylate buffer, dehydrated in alcohol, and embedded in epoxy resin. Cross-sections (1-μm thick) were cut with an ultramicrotome, mounted on glass slides, and stained with 1% toluidine blue.
Tissue sections were examined under a photomicroscope (Microphot FXA; Nikon, New York, NY). Images were captured using a camera (Megaplus, model 1.4; Eastman Kodak, Rochester, NY) connected to a frame grabber (MegaGrabber; Perceptics, Knoxville, TN) in a computer (Macintosh 8100/80 AV; Apple Computer, Cupertino, CA) using image acquisition and analysis software (Metamorph; Molecular Devices, Sunnyvale, CA).