Grading Retinal Axon Loss in Glaucoma

Whole mounts of DBA/2J retinas were used in immunohistochemistry with an antibody against neurofilaments (SMI32, Covance, Dedham, MA). Staining with this monoclonal antibody is a sensitive marker of axon loss and can be used to grade the severity of the ganglion cell loss in the retina [13 (link),64 (link)]. The fixed retinas as described above were blocked in 4% normal donkey serum and 0.1% Triton X-100 in PBS at 4 °C overnight and incubated with mouse anti-SMI32 (1:100) at 4 °C for 3 days and then visualized with a secondary antibody conjugated to tetramethyl rhodamine (1:200; Jackson Immunoresearch Laboratories, West Grove, PA). Images of each retina were taken on a BX51 microscope equipped with epifluorescence (Olympus, Center Valley, PA). Retinas were graded as showing no or early glaucoma (NOE), moderate disease (<50% axon loss), and severe disease (≥50% axon loss).