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 in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 °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.
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.
All procedures were carried out in compliance with and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Southern California. Fourteen (n = 14) Dutch-belted rabbits between 4 and 6 months of age were used in the development of this protocol. Both male and female animals were used. All animals weighed between 2.0 and 2.5 kg. All animals were singly housed. A list of recommended materials and equipment can be found in the Table of Materials.
1. Preparation
2. Initial fixation
3. Cornea and iris removal
4. Completion of fixation
5. Lens removal
6. Embedding
7. Cryosectioning
After tissue processing, a standard immunofluorescence protocol may be utilized to investigate any number of biological processes within the retina. Figure 3A–C illustrate representative fluorescence images of a retinal section obtained via confocal microscopy. The retinal section was immunostained according to a previously described protocol12.
The representative retinal sections shown in Figure 3A–C are near the optic nerve head and oriented orthogonally to the axis of the retinal vessels (that is, the section contains peripheral retina, retinal vessels, myelinated nerve fibers, and the horizontal visual streak). Sox2+ nuclei may be seen within the nerve fiber layer and inner nuclear layer. RPE65 staining demonstrates a continuous layer of retinal pigment epithelium (RPE) cells adjoining the photoreceptor layer. The fluorescence images demonstrate a uniform pigmented RPE layer without significant detachment from the overlying photoreceptor layer. A brightfield image of the retinal section further demonstrates intact retinal morphology of all layers including the photoreceptor and RPE layers (Supplemental Figure S1). Small focal detachments may be seen in the peripheral retina, likely induced during the removal of the lens or manipulation during sectioning. Fluorescence images demonstrate uniform inner and outer nuclear layers without any significant laminar disruption or distortion, suggesting minimal tissue damage during described processing.
Figure 1: Dissection of rabbit eye. (A) An example setup of necessary equipment, including forceps, curved scissors, a scalpel blade, a 100 mm dissection dish, and a dissection microscope. (B) Initial corneal incision may be made with a scalpel blade and should be parallel to the plane of the iris. (C) The cornea may be removed by making a circumferential cut with curved scissors. (D) The iris may be removed by making a circumferential cut in a similar fashion to the cornea. (E) The lens may be mobilized and removed by cutting the zonules with curved scissors. Please click here to view a larger version of this figure.
Figure 2: Embedding of rabbit eye. (A) After overnight incubation in OCT, the hyaloid membrane should appear lifted. The hyaloid canal (arrowhead) may be visible and can be used for orientation purposes. (B) For better orientation, the hyaloid membrane may be dissected to reveal the optic nerve head underneath. (C) The eye should not touch any part of the cryomold and should be sufficiently submerged without significant bubble formation. (D) Cryomolds should be placed on dry ice. The appearance at various stages of freezing is shown. (E) After freezing, the block may be sectioned in the cryostat. This block is oriented with the pupil facing rightward. OCT is visible within the eye and in close apposition to the inner and outer surface of the eye. Abbreviation: OCT = optimal cutting temperature. Please click here to view a larger version of this figure.
Figure 3: Representative fluorescence image of a whole retinal cryosection (20x magnification). (A) The RPE layer is stained with Rpe65 (red). It is continuous and in close apposition to the photoreceptor layer. The nuclei are stained with DAPI (blue). Sox2 (green) shows the location of Müller glia nuclei within the inner nuclear layer and a subset of amacrine cells in the ganglion cell layer. (B) An image of the rabbit medullary ray, a horizontal streak of myelinated retinal nerve fiber layer. (C) An image of mid-peripheral rabbit retina. Scale bars = 1,000 µm (A), 100 µm (B,C). Abbreviations: RPE = retinal pigment epithelium; DAPI = 4'6,-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Supplemental Figure S1: Representative Brightfield image of a whole retinal cryosection (20x magnification). (A) Retinal layers are intact throughout the majority of the section. Small areas of focal detachment may be seen in the far periphery of the retina. (B) An image of the rabbit medullary ray demonstrating intact morphology of all retinal layers including the photoreceptor and RPE layers. (B) An image of the mid-peripheral rabbit retina demonstrating intact morphology of all retinal layers. Scale bars = 1,000 µm (A), 100 µm (B,C). Abbreviation: RPE = retinal pigment epithelium. Please click here to download this File.
Prior to implementing the above protocol, we consistently faced difficulties with the tissue processing of rabbit eyes for IHC. We had adapted several protocols from the eyes of smaller animals such as mice but found these to lead to inadequate fixation and difficulty with tissue sectioning. There are several important considerations that allow for consistent, high-quality sections of the rabbit retina.
One consideration is the large size of the rabbit globe in comparison to other commonly used animal models such as rodents. Because the rabbit eye is similar in size to the human eye, translational studies that utilize ocular procedures commonly done in humans may be readily performed. For example, procedures involving the retina and/or the vitreous such as intravitreal injection, laser photocoagulation, vitrectomy, and surgery for retinal detachment can all be safely and easily performed in rabbits13,14,15. However, the large size of the rabbit globe requires special considerations with regard to tissue fixation and embedding procedures that make the use of protocols developed in smaller animals less effective.
Appropriate fixation depends on several factors related to the type of tissue and what the tissue will ultimately be used for. In a large eye, it is important to ensure sufficient fixative penetration to the retina. Early removal of the cornea and iris allows for a more even fixative penetration to the retina by increasing direct access to the vitreous body. Although removal of the lens may also increase fixative penetration, we have found that early removal of the lens often leads to large retinal detachments that may involve the horizontal visual streak. By removing the lens after fixation, retinal detachments are less likely to occur because of increased crosslinking within the retina, which lends additional rigidity and better toleration of the mechanical forces exerted on the retina during dissection. The type and duration of fixative are also important factors to consider, with protocols recommending anything from 4% PFA, as in this protocol, to alternatives such as Davidson's fixative or those containing various concentrations of glutaraldehyde. Although other fixatives have been reported to result in less tissue shrinking and may better preserve certain epitopes, we have found that 4% PFA results in excellent IHC staining if used between 16 h and 24 h at 4 °C. It is important to note that minor tissue shrinkage can result in a wave-like appearance of the outer nuclear layer in some areas, although this appearance will likely not affect major results.
Another important point to consider when preparing a larger eye such as a rabbit eye for IHC is the embedding procedure. OCT is a popular embedding material for use in IHC as it preserves tissue morphology and the antigenicity of a wide range of epitopes within the tissue, making it compatible with most IHC protocols and reagents. To preserve retinal morphology, it is important that OCT approximates the outside of the globe (the sclera side) and completely infiltrates and approximates the inside of the globe (the retina side). Incubating the eye in OCT overnight is one easy method to ensure this occurs. This allows sufficient time for the OCT to infiltrate the vitreous and results in detachment of the posterior hyaloid membrane away from the retina, allowing OCT to directly line the innermost surface of the retina. It is likely that OCT liquefies or otherwise causes contraction of the vitreous just as in posterior vitreous detachment.
The protocol described here provides a detailed and readily adaptable method for obtaining consistently high-quality retinal sections from whole rabbit eyes for use in IHC. Although many protocols exist for smaller animals such as rodents, very few protocols exist for processing rabbit retinas. Even in smaller animals, it is often very difficult to reliably obtain whole retinal sections without significant artifact formation. This results in the loss of valuable time and resources, as well as negatively affects the interpretation of results. This method minimizes tissue damage and cryo section loss. Furthermore, this method may easily be adapted for use in other larger mammals such as pigs, cows, or monkeys. Because this protocol requires careful dissection involving the lens and vitreous body, it is less applicable for use in smaller animals such as rodents. Future studies should investigate alternative methods for use in smaller eyes. In addition, future studies should investigate utilizing different fixation or embedding techniques that may further optimize results. Other modifications may be made to further minimize artifacts, such as the use of glue to stabilize the eye cup.
The authors have nothing to disclose.
Thanks to Rosanna Calderon, Dominic Shayler, and Rosa Sierra for technical advice. This study was supported in part by an unrestricted grant to the Department of Ophthalmology at the USC Keck School of Medicine from Research to Prevent Blindness (AN), NIH K08EY030924 (AN), the Las Madrinas Endowment in Experimental Therapeutics for Ophthalmology (AN), a Research to Prevent Blindness Career Development Award (AN), Knights Templar Eye Foundation Endowment (AN), and the Edward N. and Della L. Thome Memorial Foundation (AN, KG).
100 mm culture dish | Corning | 353025 | Used for dissection (steps 1.3, 3, and 5) |
50 mL tube | Genesee Scientific | 28-106 | For fixation and cryoprotection (step 1) |
Cryostat | Leica | CM1850 | For cryosectioning (step 7) |
Curved scissors | Fine Science Tools | 91500-09 | Used for dissection (steps 1.3, 3, and 5) |
DAPI | Fisher Scientific | D3571 | Diluted 1:1,000 in blocking buffer |
Dissection microscope | Zeiss | Stemi 2000-C | Used for dissection (steps 1.3, 3, and 5) |
Donkey anti-Goat 488 | Fisher Scientific | A-11055 | Diluted 1:1,000 in blocking buffer |
Donkey anti-Mouse 555 | Fisher Scientific | A-31570 | Diluted 1:1,000 in blocking buffer |
Forceps | Fine Science Tools | 91150-20 | Used for dissection (steps 1.3, 3, and 5) |
Glass Slide Cover | VWR | 48404-453 | For cryosectioning (step 7) |
Goat anti-SOX2 | R&D Systems | AF2018 | Diluted 1:100 in blocking buffer |
High-profile disposable cryostat blades | Leica Microsystems Inc. | 14035838926 | For cryosectioning (step 7) |
Kimwipe | Fisher Scientific | 06-666-A | Used to wipe away excess PBS or OCT (steps 3 and 6) |
Mouse anti-RPE65 | Novus Bio | NB100-355SS | Diluted 1:100 in blocking buffer |
OmniPur Sucrose | Millipore | 167117 | Used for cryoprotectant (step 1.2) |
Paraformaldehyde 20% solution | Electron Microscopy Sciences | 15713 | Used as tissue fixative (diluted to 4% in step 1.1) |
Peel-A-Away Disposable Embedding Mold (22x22x20 mm Deep) | Polysciences, Inc. | 18646A | Used as embedding mold (step 6) |
Phosphate buffered saline, 1x | Corning | 21-030-CV | Used in preparation of fixative (step 1.1) and cryoprotectant (step 1.2) |
Scalpel blade no. 15 | Feather | 08-916-5D | Used for dissection (steps 1.3, 3, and 5) |
Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | For cryosectioning (step 7) |
Tissue-Tek O.C.T. Compound | Sakura | 4583 | Used as embedding media (step 6) |