This study investigates retinal ganglion cell (RGC) degeneration due to increased intraocular pressure (IOP) in a rat model. The methodology includes immunohistochemical techniques to visualize RGCs and assess cell density in damaged retinas.
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 the tissue by washing with a 0.5% non-ionic surfactant diluted in PBS. Then, incubate the tissue in blocking solution for one hour at room temperature with gentle shaking. During the incubation, prepare BRM 3A primary antibody solution and store at 4 degree celsius.
After tissue blocking, incubate the retinas in 200 microliters of primary antibody solution at 4 degrees celsius for 72 hours with gentle shaking. Next, wash the tissue with PBS. Incubate the tissue in 200 microliters of the secondary antibody solution for two hours at room temperature, and then with nuclear counterstain solution for 10 minutes for fluorescent nuclei staining.
After three PBS washes, transfer the retina onto a glass microscope slide using two small brushes, maintaining the vitreous side up. Position the dorsal retinal quadrant up on the microscope slide. Finally, apply 200 microliters of anti-fade mounting medium on a glass coverslip and place it onto the flat mounted retina for microscopic analysis. Under a confocal epi-fluorescence microscope using a 40 times magnifying objective, examine the flat mounts to estimate retinal ganglion cells.
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