Quantification of Retinal Ganglion Cell Somas Following Cauterization-Induced Ocular Hypertension

Take a chemically-fixed retina isolated from a rat's eye.

The eye exhibits heat-induced damage to the limbal vascular plexus, a network of blood vessels surrounding the cornea.

This damage blocks fluid drainage, increasing intraocular pressure (IOP) and causing retinal ganglion cell (RGC) degeneration.

Treat the retina with a permeabilization solution to permeabilize the cell and nuclear membranes.

Apply a blocking solution to prevent non-specific antibody binding.

Remove the blocking solution.

Introduce primary antibodies that target a transcription factor predominantly expressed in RGCs.

Wash to remove unbound antibodies.

Introduce fluorophore-conjugated secondary antibodies that bind to the primary antibodies.

Wash to remove unbound antibodies.

Counterstain the nuclei with a DNA-binding dye.

Wash the retina and mount it on a glass slide using an appropriate mounting medium.

Visualize the retina under an epifluorescence microscope.

A reduced cell density in the damaged eye compared to the control confirms RGC degeneration due to increased IOP.