After the treated eyes were exposed to elevated IOP for a certain period, both eyes of each animal were removed and prepared for immunohistochemical study. Tissue preparation followed previously developed procedures.
28,29 Briefly, both eyes of an anesthetized animal were removed, and the animal was euthanatized. For each eye, an eye cup of 5 mm diameter that included the optic nerve was excised and placed in a dish of warm (33–35°C) oxygenated physiologic solution. After it was dissected from the retinal pigment epithelium and choroid, the retina was placed on a membrane with the photoreceptor side against the membrane, fixed in 4% paraformaldehyde for 30 minutes at room temperature, and rinsed thoroughly in phosphate-buffered saline (PBS). The tissue was then removed from the membrane for further immunohistochemical staining.
Orientation of retinas was documented by first marking the caudal side of the eye in situ with a skin marker and then, after dissection, cutting a notch into the eye cup and retinal edge at the marked position.
Three major cytoskeleton components of axons were studied by multiply labeling a whole-mounted retina with phalloidin to stain F-actin, anti-β-tubulin monoclonal antibody to mark the MTs, and anti-neurofilament antibody to label the NFs. The nuclei in the inner retina were also identified by 4′,6-diamidino-2-phenylindole (DAPI) fluorescent counterstain. The tissue was permeabilized in PBS containing 0.8% TritonX-100 for 1 hour followed by incubation in blocking serum (5% goat serum and 0.8% TritonX-100) for 1 hour at room temperature. The tissue was transferred into a primary antibody solution (1:500, rabbit anti-neurofilament 200 kDa Sigma) at 4°C overnight. After the tissue was washed in PBS (three changes of 10 minutes each), it was incubated in a mixed solution of the secondary antibody (1:250, AlexaFluor 647 goat anti-rabbit IgG; Invitrogen, Carlsbad, CA) and anti-β-tubulin antibody (1:100, Cy3 conjugated; Sigma-Aldrich, St. Louis, MO) overnight at 4°C. After it was again washed in PBS, the tissue was transferred into a solution of phalloidin (1:100, AlexaFluor 488 phalloidin; Invitrogen) for 1 hour at room temperature. After a rinse in PBS, it was incubated in DAPI (FluoroPure grade; Invitrogen-Molecular Probes, Eugene, OR) for 30 minutes in subdued lighting. The stained retina was rinsed again and mounted on a glass slide with an antifade mounting medium (Vectashield; Vector Laboratories, Inc., Burlingame, CA). The retina was stored at 4°C for confocal microscopy imaging.
A series of control experiments were performed and confirmed that the retinas had weak autofluorescence, the secondary antibody specifically bound to the primary antibody for NF labeling, and there were unnoticeable spectral bleed-through signals between fluorescence detectors.