Encyclopedia of Experiments: Biology
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Transcript
- A zebrafish eye comprises cornea, sclera, lens, and retina. The cornea is a transparent tissue that allows light to enter into the eye. The sclera is an opaque tissue that is continuous with the cornea, forming an outer protective layer. The lens is a spherical structure that focuses light onto the retina, which is a light sensitive layer of tissue that converts light into a neural signal.
To dissect the eye, fill a Petri dish with silicone and let it set. Now, place an anesthetized fish in the dish and excise the eyes using a micro-scissor. Transfer the eyes to a plate filled with PBS to maintain a constant pH in the tissue and orient the eye with the posterior side facing up. Hold the eye with forceps and carefully make three radial cuts through the sclera and retina.
Next, fold the tissue out to reveal the lens. Now, position the eye with the anterior side facing up and remove the sclera and retina. Scoop out the lens from the dissected eye and remove any remaining tissue around it. In the following protocol, we will perform larval and adult zebrafish lense dissections to analyze lens morphology.
After confirmation of sufficient sedation with tricaine, use microdissection scissors to immediately excise both eyes from either an adult or larval zebrafish, and place both eyes into a 35-millimeter custom-made Petri dish with a silicone dissection mold filled with PBS. To harvest the lens from an adult zebrafish, place the eye posterior side up and insert forceps at a less than 45 degree angle through the optic disk. Use dissection scissors to make two or three radial incisions through the retina and sclera from the optic disk to the ciliary zone in the immobilized eye and peel back the retina and sclera like flower petals. Invert the eye, cornea side up, and use the flat side of the scissors to immobilize the lens indirectly via manipulation of the sclera and cornea while using forceps to pull away the retina and attached tissues. Then carefully trim away any excess tissue from the lens.
For larval zebrafish lens harvest, plays a larval eye posterior side up onto the flat part of a silicone dish of PBS. And use a sharpened tungsten needle to make radio cuts through the retina and sclera while immobilizing the eye with another needle or forceps. Then gently scoop the lens from the dissociated eye with the blunt side of a custom-made tungsten wire needle and carefully pull away the attached tissue.
