Enucleated eyes (2 rabbits from each group) were fixed in 4% paraformaldehyde for 2 hours. After the anterior segment of the globe was removed, the eyecups were fixed further in paraformaldehyde overnight and then transferred to 25% sucrose solution for an additional 24 hours. Blocks were cut from the posterior eyecup 4 disc diameters below the optic nerve head and then embedded in optimal cutting temperature compound with 2-methylbutane and dry ice. Cryosections (5-μm thick) were prepared at −20°C, fixed in cold methanol for 15 minutes, rinsed in 1× PBS for 5 minutes, and incubated with 10% goat serum for 45 minutes at room temperature.
The primary antibodies were anti–glial fibrillary acidic protein (GFAP) antibody (mouse IgG, 1:200; BD PharMingen, Franklin Lakes, NJ) as a marker for glial cells and anti–acrolein antibody (mouse IgG, 1:100; Genox Corporation, Baltimore, MD) as a marker for oxidative protein modification (FDP-lysine derivatives). Incubation with either primary antibody was performed for 1 hour at 37°C. Sections then were washed with 1× PBS three times and incubated with either FITC (anti-mouse IgG, 1:200; Abcam, Cambridge, MA)– or Texas Red (anti-mouse IgG, 1:200; American Qualex, San Clemente, CA)–conjugated secondary antibody at room temperature for 1 hour. Retinal sections were washed for 30 minutes with 0.1 M PBS and coverslipped with mounting medium (Vectashield; Vector Laboratories Inc., Burlingame, CA) containing 4,6-diamidino-2-phenylindole (DAPI). Sections were then analyzed using a confocal microscope (LSM 510; Carl Zeiss Inc., Thornwood, NY). FITC staining was captured using the 488-nm argon laser. Texas Red staining was captured using a 543 nm HeNe laser. DAPI staining was captured using 800 nm titanium sapphire laser. Immunofluorescence images were processed using Zeiss software (LSM-PC; Carl Zeiss Inc.). Corresponding negative controls were prepared by substitution of the primary antibody with 10% normal goat serum in PBS.