ocular tissue embedding and cryosectioning in optimal cutting temperature compound for fluorescence imaging 

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

The retina is composed of several layers of specialized cells within the eye that together work to convert light into neural signals. Because the retina plays a critical role in vision, understanding its structure and function can provide valuable insights into some of the most common causes of vision loss such as macular degeneration and diabetic retinopathy, among others.
Rabbits serve as a convenient animal model in retinal research as they offer several advantages compared to other models. Rabbit eyes are relatively similar in anatomy to human eyes1,2. For example, rabbits have an area of increased photoreceptor density, known as the horizontal visual streak, that is analogous to the fovea in humans. Other commonly used animal models, such as rodents, do not have an anatomic equivalent. In addition, compared to rodents, the retinal vasculature in rabbits is fairly similar to that in humans. Rabbit eyes are relatively large as well. This makes them particularly suitable for studies that involve drug administration or surgical intervention within the vitreous or retina that may otherwise be difficult or impossible in a smaller eye3.
Immunohistochemistry (IHC) is a widely used technique to study the localization of antigens within a tissue and has broad applications in retinal research4,5,6. Because the retina is a delicate structure, obtaining useful results via IHC requires careful tissue processing. Retinal detachment and other tissue artifacts such as retinal breaks or folds commonly occur during processing and may interfere with the interpretation of results. Successful processing depends on a variety of factors, including tissue manipulation, type and duration of fixation, type of embedding media, and sectioning techniques7,8,9,10. Despite the advantages of using rabbits as an animal model in retinal research, very few protocols describing successful tissue processing of the rabbit retina exist. This paper describes a reliable method for obtaining high-quality retinal sections from whole rabbit eyes for use in IHC.

Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models

Obtaining High-Quality Cryosections of Whole Rabbit Eye 

Our research focuses on novel approaches to regenerating the retinal pigment epithelium. Dysfunction of the retinal pigment epithelium is thought to play a central role in the pathophysiology of many eye diseases, such as macular degeneration, so regenerating this layer of cells is attractive from a therapeutic standpoint. Visualizing the structural anatomy of the retina via immunohistochemistry has wide applications in retinal research.In our laboratory, we found obtaining useful results from rabbit eyes, difficult due to tissue artifacts like retinal attachments and retinal folds. Despite these challenges, there are very few protocols existing that describe successful tissue processing of rabbit retina. This protocol makes several important modifications to standard tissue processing techniques that result in excellent tissue sections of the rabbit retina.This study may allow researchers to obtain better results from their research that involves the use of immunohistochemistry in rabbit models.

Ocular Tissue Embedding and Cryosectioning in Optimal Cutting Temperature Compound for Fluorescence Imaging 

After cryo protection of the rabbit eye is complete, transfer it from a 30%sucrose solution to a 100 millimeter dissection dish. Under a dissecting microscope, carefully flip the eye to remove excess sucrose solution from the inner part of the eye. Using a wipe, gently wipe away excess sucrose solution from the outer part of the eye, fill a disposable embedding mold with optimal cutting temperature or OCT compound to a depth of 0.5 to one centimeter.Place the eye in the embedding mold facing upward and slowly fill the inner part of the eye with OCT compound, avoiding bubble formation. Once the eye is emerged in the OCT compound, and does not touch the inner surfaces of the mold, wrap the embedding mold in paraffin and store it at four degrees Celsius overnight. The next day, place the embedding mold in a dissection dish.If possible, gently remove the OCT compound from the inside of the eye with a wipe. Transfer the eye to a new embedding mold filled with fresh OCT compound. Then fill the mold as previously done to ensure the final embedding medium is free of debris or imperfections.Label the mold for orientation purposes. Position the eye upward in the cryo mold, so the OCT compound lines the inner retina. Use the optic nerve head as a landmark for determining the superior part of the eye Place the labeled embedding mold on dry ice, ensuring that the eye is not touching the inner surfaces of the mold or exposed to air.Mount the block on the cryostat with the superior part of the eye facing up and the inferior part down. With the remaining cornea and iris facing to the right or left. Position a fresh blade parallel to the vertical axis of the eye to section it.After cutting the sections and drying overnight, place a slide in a freezer at 80 degrees Celsius. The fluorescence images of a whole retinal cryosection of rabbit eye demonstrated a uniform pigmented retinal pigment epithelium layer without significant detachment from the overlying photoreceptor layer. A bright-field image of the retinal section further demonstrates intact retinal morphology of all layers.

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Research

JoVE Journal

Biology

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

Whole Rabbit Eye Dissection and Ocular Tissue Preparation for Histological Analysis

After transconjunctival enucleation of the rabbit eye, immediately place in 50 milliliters of 4%paraformaldehyde and PBS on ice. After 15 minutes, transfer the eye to a 100 millimeter dissection dish and fill the dish with PBS to prevent the eye from drying out. Under a dissection microscope, Using a surgical blade, start making an incision one millimeter anterior to the limbus, keeping it parallel to the iris plane.Insert curved scissors into the initial incision and continue cutting circumferentially, maintaining a one millimeter distance from the limbus. Then remove the cornea with forceps and gently wipe away PBS With a laboratory wipe. Place the cornea in 30%sucrose solution at room temperature for cryoprotection.Make an initial cut with curved scissors through the iris. Then cut circumferentially around the eye, similar to the cornea removal, to separate the iris from the rest of the tissue. Remove the iris with forceps and gently wipe away excess PBS with the laboratory wipe.Place the iris in 30%sucrose solution at room temperature for cryoprotection. After removing the cornea and iris, place the eye in 50 milliliters of 4%paraformaldehyde and put it on a shaker for gentle agitation. Leave the eye in 4%paraformaldehyde at four degrees Celsius overnight.The next day, place the eye in a dissection dish and fill the dish with PBS. Under the dissection microscope, use forceps to carefully grasp the anterior lens capsule. Then carefully insert the scissors into the posterior chamber, orienting the blades posteriorly along the lens.Make an initial cut through the lens zonules with curved scissors. Continue making small cuts in a circumferential pattern through the lens zonules. To confirm lens removal, gently grasp the anterior lens capsule with forceps and attempt to lift it.Upon separation, place the lens in 30%sucrose solution at room temperature for cryoprotection. Place the eye in 50 milliliters of 30%sucrose and PBS at four degrees Celsius for 24 to 48 hours for cryoprotection. To expedite cryoprotection, replace the initial sucrose solution with a fresh one after eight to 12 hours.