JoVE Encyclopedia of Experiments
Neuroscience
0 views • 3:06 min • April 28th, 2025
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.
After isolating the retinas, transfer them into a 24-well culture plate containing 1 milliliter PBS, keeping the inner retina facing up. Permeabilize t
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