After euthanizing the mouse using cervical dislocation for enucleation and eyeball processing, mark the edge of the cornea on the dorsal side of the eyeball using a blue marker to ensure orientation during dissection. Using scissors, remove residual muscle tissues attached to the eyeball. Then transfer the enucleated eyeball into a two milliliter round bottom micro centrifuge tube filled with PBS.
Now, place the eyeball into another two milliliter tube containing freshly prepared 4%paraformaldehyde for fixation at four degrees Celsius for 10 minutes. Under a dissecting microscope, use fine forceps to make a small incision on the cornea. Place the eyeball back into the fixative and incubate at four degrees Celsius for three hours to preserve tissue morphology.
To remove the cornea and lens, using fine surgical scissors make a small incision of approximately one millimeter around the corneal limbus, the border between the cornea and the sclera. Carefully cut around the circumference of the cornea, excising it entirely to expose the underlying structures. Next, using fine forceps, gently grasp and carefully lift the lens from the surrounding tissues to avoid damaging the retina.
For scleral incision, cut a precise slit in the sclera with fine ophthalmic scissors to facilitate subsequent dissection. Next, for retinal separation, insert toothed forceps into the incision between the sclera and the retina. Gently grasp the edge of the sclera, avoiding excessive force to prevent tearing the tissue.
Gradually enlarge the incision by moving the forceps along the boundary between the sclera and the retina. Use the tooth part of the forceps to carefully separate the sclera from the retina without damaging it. Now, using sharp scissors, divide the retina into four roughly equal sized flaps.
Gently unfold the dissected retina with a soft bristle brush and meticulously removed debris from its surface.
