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).

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&#160;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
<ol>
	<li>Fixative preparation.
	<ol>
		<li>Prepare 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). At a minimum, 50 mL of fixative is required per eye.</li>
	</ol>
	</li>
	<li>Cryoprotective agent preparation.
	<ol>
		<li>Prepare 30% weight by volume (w/v) sucrose in PBS. Place on a shaker for at least 15 min to ensure that the sucrose is completely dissolved. At a minimum, 50 mL of cryoprotective agent is required per eye.</li>
	</ol>
	</li>
	<li>Dissection instrument preparation.
	<ol>
		<li>Gather two pairs of forceps, one pair of curved scissors, one scalpel blade, and a 100 mm dissection dish and place them near a dissection microscope. An example setup is shown in Figure 1A.</li>
	</ol>
	</li>
</ol>
2. Initial fixation
<ol>
	<li>Following trans-conjunctival enucleation11, immediately place the eye in at least 50 mL of 4% PFA in PBS on ice. Ensure that the eye is completely submerged. If air bubbles collect on the outer surface of the eye, remove them by gently shaking.</li>
</ol>
3. Cornea and iris removal
<ol>
	<li>After 15 min, remove the eye from 4% PFA in PBS and place it in a 100 mm dissection dish under a dissecting microscope. Fill the dish with PBS to prevent the eye from drying out.</li>
	<li>Remove the cornea.
	<ol>
		<li>Begin by creating an incision with a surgical blade 1 mm anterior to the limbus, parallel to the iris plane to avoid accidentally puncturing underlying structures (Figure 1B).</li>
		<li>Insert curved scissors into the initial incision and continue cutting circumferentially while remaining 1 mm from the limbus. Use forceps to stabilize the eye while cutting (Figure 1C).</li>
		<li>Once the circumferential cut is complete, remove the cornea with forceps and gently wipe away excess PBS with a laboratory wipe. Place the cornea in 30% sucrose solution at room temperature (RT) for cryoprotection or discard. Cryoprotection is complete when the cornea sinks, which typically takes less than 1 h.</li>
	</ol>
	</li>
	<li>Remove the iris.
	<ol>
		<li>Begin by making an initial cut with curved scissors through the iris. Completely separate the iris from the rest of the tissue by cutting circumferentially similar to cornea removal (Figure 1D).</li>
		<li>Once the circumferential cut is complete, remove the iris with forceps and gently wipe away excess PBS with a laboratory wipe. Place the iris in 30% sucrose solution at RT for cryoprotection or discard as above. Cryoprotection is complete when the iris sinks, which typically takes less than 30 min.</li>
	</ol>
	</li>
</ol>
4. Completion of fixation
<ol>
	<li>With the cornea and iris removed, place the eye in at least 50 mL of freshly prepared 4% PFA in PBS and put it on a shaker for gentle agitation. Ensure the eye remains submerged during agitation.</li>
	<li>Keep the eye in 4% PFA in PBS at 4 &#176;C overnight. 
	&#8203;NOTE: We have found that 4% PFA results in excellent IHC staining if used between 16 h and 24 h at 4 &#176;C, with longer fixation durations resulting in increased nonspecific background staining and epitope masking.</li>
</ol>
5. Lens removal
<ol>
	<li>The following morning, remove the eye from 4% PFA in PBS and place it in a 100 mm dissection dish under a dissecting microscope. Fill the dish with PBS to prevent the eye from drying out.</li>
	<li>Remove the lens.
	<ol>
		<li>Begin by carefully grasping the anterior lens capsule with forceps. 
		NOTE: Be extremely careful not to forcefully push or pull on the lens with the forceps as this can create a retinal detachment.</li>
		<li>Carefully insert the scissors into the posterior chamber with the blades curved posteriorly along the lens. Make an initial cut through the lens zonules with curved scissors. 
		NOTE: Be extremely careful not to forcefully push or pull on the zonules with the scissors as this can create a retinal detachment (Figure 1E).</li>
		<li>Continue to make small cuts through the lens zonules in a circumferential pattern. Make three to five cuts per clock hour.</li>
		<li>To check if the lens is separated from the rest of the tissue, gently grasp the anterior lens capsule with forceps and attempt to lift it gently. If the lens does not immediately lift, do not attempt to pull more as this can create a retinal detachment. Instead, make more cuts through the remaining lens zonules and reattempt.</li>
		<li>Once separated from the rest of the tissue, place the lens in 30% sucrose solution at RT for cryoprotection or discard as above. Cryoprotection is complete when the lens sinks, which typically takes a few hours.</li>
	</ol>
	</li>
	<li>Place the eye in at least 50 mL of 30% sucrose in PBS at 4 &#176;C for cryoprotection. Cryoprotection is complete when the eye sinks, which typically takes 24-48 h. To expedite cryoprotection, exchange the initial sucrose solution with a fresh one after 8-12 h and/or keep the eye in sucrose solution at RT.</li>
</ol>
6. Embedding
<ol>
	<li>Once cryoprotection is complete, remove the eye from 30% sucrose solution and place it in a 100 mm dissection dish under a dissecting microscope. Carefully flip the eye so it faces downward to remove excess sucrose solution from the inner part of the eye. 
	NOTE: Do not attempt to remove excess solution from the inner part of the eye with a laboratory wipe as the wipe can stick to the vitreous body and result in a retinal detachment.</li>
	<li>With the eye facing downward, gently wipe away excess sucrose solution from the outer part of the eye using a wipe.</li>
	<li>Fill a disposable embedding mold with optimal cutting temperature (OCT) compound to a depth of 0.5-1.0 cm. 
	NOTE: This base ensures the eye will not touch the bottom of the mold, which interferes with cryosectioning later.</li>
	<li>Place the eye in the disposable embedding mold facing upward. Slowly fill the inner part of the eye with OCT taking special care to avoid bubble formation within the eye. If bubbles do form, carefully remove them or push them to the edge of the mold using forceps.</li>
	<li>Finish filling the disposable embedding mold with OCT. Ensure the eye is completely submerged in OCT and is not touching the inner surfaces of the mold or exposed to the air. Wrap the disposable embedding mold containing the eye in OCT in parafilm; place at 4 &#176;C overnight. 
	NOTE: In our experience, shorter incubation periods do not allow OCT to completely infiltrate the vitreous. As a result, a layer of sucrose often remains in between the retina and OCT. When this occurs, the retina often detaches or completely tears away from the rest of the tissue during cryosectioning as frozen sucrose likely provides less structural support than frozen OCT, making high-quality sections difficult or even impossible to obtain.</li>
	<li>The following morning, place the embedding mold containing the eye in OCT in a 100 mm dissection dish under a dissecting microscope. Confirm that the posterior hyaloid membrane appears lifted upward from the inner retina. 
	NOTE: This upward lifting may obscure the view of the retina, making orientation difficult to determine.</li>
	<li>To better determine the orientation of the eye, carefully grasp the posterior hyaloid membrane anteriorly and attempt to peel it away with forceps to expose the underlying retina while in OCT. 
	NOTE: At times, the hyaloid canal may be visible, which attaches to the optic nerve head and can be used for orientation purposes since the optic nerve head is located in the superior portion of the retina (Figure 2A). It is not necessary to completely remove the posterior hyaloid membrane as OCT has already infiltrated the vitreous body to line the inner retina and any remaining membrane will not affect cryosectioning (Figure 2B). The posterior hyaloid membrane should only be removed for orientation, if necessary. Other options for determining orientation include placing a suture or other type of mark prior to enucleation.</li>
	<li>Transfer the eye to a new embedding mold with new OCT filled to a depth of 0.5-1.0 cm and then fill with new OCT as in steps 6.3-6.5&#160;to ensure that the final embedding medium is free of debris and/or other imperfections that may have accumulated overnight. If possible, gently remove OCT with a wipe from the inside of the eye as well, although this is not necessary.</li>
	<li>Label the mold for orientation purposes (Figure 2C). Orienting the eye with the eye facing upward in the cryo mold ensures that OCT will line the inner retina. The hyaloid canal, which attaches to the optic nerve head, is an important landmark for determining the superior part of the eye (Figure 2A).</li>
	<li>Place the labeled embedding mold containing the eye in OCT on top of dry ice. Ensure that the eye is not touching any of the inner surfaces of the embedding mold or exposed to air. Ensure the mold is directly touching the dry ice to speed up the freezing process (Figure 2D).</li>
	<li>Transfer the embedded eye to a -80 &#176;C freezer or directly to the cryostat for sectioning (Figure 2E).</li>
</ol>
7. Cryosectioning
<ol>
	<li>Mount the block with the superior part of the eye facing superiorly and the inferior part of the eye facing inferiorly, with the remaining cornea and iris facing to the right (as in Figure 2E) or left (not shown). Alternatively, mount the block with a different orientation, as desired. If necessary, break the mold to mobilize the block by carefully breaking off each side of the mold one at a time and marking the block directly to not lose the orientation of the eye.</li>
	<li>Allow the block to equilibrate to the temperature within the cryostat for 30-60 min; -20 &#176;C is a good starting point.</li>
	<li>Once mounted, place a fresh blade at the desired angle, typically parallel to the vertical axis of the eye.</li>
	<li>Set the section thickness as desired: 10-20 &#181;m thick sections are a good starting point. An example of a 14 &#181;m thick section is demonstrated in Figure 3A-C.
	<ol>
		<li>In a slow and controlled manner, begin sectioning through the block. Ensure the blade is cutting the block parallel to the vertical axis of the eye. If not, adjust the angle the blade contacts the block or stage orientation accordingly.</li>
		<li>While sectioning the block, prevent wrinkling or curling of the section with a brush. Quickly flip the section over and apply gentle pressure downward with a long-haired brush to flatten the section and minimize wrinkling or curling. Fashion a separate brush with a pointed tip to help manipulation of the section.</li>
	</ol>
	</li>
	<li>Attach the section to a charged glass slide by slowly moving the slide facing downward toward the section. Prevent the section from folding on itself or otherwise distorting itself during attachment by gently rolling the glass slide over the section. 
	NOTE: Do not hover the slide directly over the section or press it against the section as this can damage or distort the section.</li>
	<li>Allow the slides to dry overnight before transferring to a -80 &#176;C freezer or proceeding directly to a standard immunofluorescence protocol.</li>
</ol>

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

<ol>
	<li>Peiffer, R. L., Pohm-Thorsen, L., Corcoran, K., Manning, P. J., Ringler, D. H., Newcomer, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+19-Models+in+ophthalmology+and+vision+research.">Chapter 19-Models in ophthalmology and vision research.</a> American College of Laboratory Animal Medicine, The Biology of the Laboratory Rabbit. , 409-433 (1994).</li><li>Davis, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+and+histology+of+the+eye+and+orbit+of+the+rabbit.">The anatomy and histology of the eye and orbit of the rabbit.</a> Trans Am Ophthalmol Soc. 27, 400.2-441 (1929).</li><li>Zernii, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+models+of+ocular+diseases:+New+relevance+for+classical+approaches.">Rabbit models of ocular diseases: New relevance for classical approaches.</a> CNS &amp; Neurological Disorders Drug Targets. 15 (3), 267-291 (2016).</li><li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labelled+antigens+and+antibodies.">Labelled antigens and antibodies.</a> Annu Rev Microbiol. 8, 333-352 (1954).</li><li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+antibodies+as+histochemical+tools.">Fluorescent antibodies as histochemical tools.</a> Fed Proc. 10 (2), 558-559 (1951).</li><li>Coons, A. H., Kaplan, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+antigen+in+tissue+cells;+improvements+in+a+method+for+the+detection+of+antigen+by+means+of+fluorescent+antibody.">Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody.</a> J Exp Med. 91 (1), 1-13 (1950).</li><li>Pang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-by-step+preparation+of+mouse+eye+sections+for+routine+histology,+immunofluorescence,+and+RNA+in+situ+hybridization+multiplexing.">Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing.</a> STAR Protoc. 2 (4), 100879 (2021).</li><li>Sorden, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+background+and+procedure-related+microscopic+findings+and+common+artifacts+in+ocular+tissues+of+laboratory+animals+in+ocular+studies.">Spontaneous background and procedure-related microscopic findings and common artifacts in ocular tissues of laboratory animals in ocular studies.</a> Toxicol Pathol. 49 (3), 569-580 (2021).</li><li>Margo, C. E., Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+of+whole+eyes:+the+role+of+fixative+osmolarity+in+the+production+of+tissue+artifact.">Fixation of whole eyes: the role of fixative osmolarity in the production of tissue artifact.</a> Graefes Arch Clin Exp Ophthalmol. 233 (6), 366-370 (1995).</li><li>Chatterjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artefacts+in+histopathology.">Artefacts in histopathology.</a> J Oral Maxillofac Pathol. 18 (Suppl 1), S111-S116 (2014).</li><li>Mitchell, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+in+companion+animals.">Enucleation in companion animals.</a> Ir Vet J. 61 (2), 108-114 (2008).</li><li>Kastan, N. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+improved+inhibitor+of+Lats+kinases+to+promote+regeneration+of+mammalian+organs.">Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs.</a> Proc Natl Acad Sci U S A. 119 (28), e2206113119 (2022).</li><li>Baiza-Dur&#225;n, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+tolerability+evaluation+after+repeated+intravitreal+injections+of+a+humanized+anti-VEGF-A+monoclonal+antibody+(PRO-169)+versus+ranibizumab+in+New+Zealand+white+rabbits.">Safety and tolerability evaluation after repeated intravitreal injections of a humanized anti-VEGF-A monoclonal antibody (PRO-169) versus ranibizumab in New Zealand white rabbits.</a> Int J Retina Vitreous. 6, 32 (2020).</li><li>Yu, D. Y., Cringle, S. J., Su, E., Yu, P. K., Humayun, M. S., Dorin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+changes+in+intraretinal+oxygen+distribution+in+pigmented+rabbits.">Laser-induced changes in intraretinal oxygen distribution in pigmented rabbits.</a> Invest Ophthalmol Vis Sci. 46 (3), 988-999 (2005).</li><li>Faude, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+artificial+retinal+detachment+for+macular+translocation+surgery+tested+in+rabbit.">Facilitation of artificial retinal detachment for macular translocation surgery tested in rabbit.</a> Invest Ophthalmol Vis Sci. 42 (6), 1328-1337 (2001).</li></ol>

The authors have no conflicts of interest to declare.

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 use...

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.

<table><tbody><tr><td>100 mm culture dish</td><td>Corning</td><td>353025</td><td>Used for dissection (steps 1.3, 3, and 5)</td></tr><tr><td>50 mL tube</td><td>Genesee Scientific</td><td>28-106</td><td>For fixation and cryoprotection (step 1)</td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1850</td><td>For cryosectioning (step 7)</td></tr><tr><td>Curved scissors</td><td>Fine Science Tools</td><td>91500-09</td><td>Used for dissection (steps 1.3, 3, and 5)</td></tr><tr><td>DAPI</td><td>Fisher Scientific</td><td>D3571</td><td>Diluted 1:1,000 in blocking buffer</td></tr><tr><td>Dissection microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td>Used for dissection (steps 1.3, 3, and 5)</td></tr><tr><td>Donkey anti-Goat 488</td><td>Fisher Scientific</td><td>A-11055</td><td>Diluted 1:1,000 in blocking buffer</td></tr><tr><td>Donkey anti-Mouse 555</td><td>Fisher Scientific</td><td>A-31570</td><td>Diluted 1:1,000 in blocking buffer</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>91150-20</td><td>Used for dissection (steps 1.3, 3, and 5)</td></tr><tr><td>Glass Slide Cover</td><td>VWR</td><td>48404-453</td><td>For cryosectioning (step 7)</td></tr><tr><td>Goat anti-SOX2</td><td>R&amp;amp;D Systems</td><td>AF2018</td><td>Diluted 1:100 in blocking buffer</td></tr><tr><td>High-profile disposable cryostat blades</td><td>Leica Microsystems Inc.</td><td>14035838926</td><td>For cryosectioning (step 7)</td></tr><tr><td>Kimwipe</td><td>Fisher Scientific</td><td>06-666-A</td><td>Used to wipe away excess PBS or OCT (steps 3 and 6)</td></tr><tr><td>Mouse anti-RPE65</td><td>Novus Bio</td><td>NB100-355SS</td><td>Diluted 1:100 in blocking buffer</td></tr><tr><td>OmniPur Sucrose</td><td>Millipore</td><td>167117</td><td>Used for cryoprotectant (step 1.2)</td></tr><tr><td>Paraformaldehyde 20% solution</td><td>Electron Microscopy Sciences</td><td>15713</td><td>Used as tissue fixative (diluted to 4% in step 1.1)</td></tr><tr><td>Peel-A-Away Disposable Embedding Mold (22x22x20 mm Deep)</td><td>Polysciences, Inc.</td><td>18646A</td><td>Used as embedding mold (step 6)</td></tr><tr><td>Phosphate buffered saline, 1x</td><td>Corning</td><td>21-030-CV</td><td>Used in preparation of fixative (step 1.1) and cryoprotectant (step 1.2)</td></tr><tr><td>Scalpel blade no. 15</td><td>Feather</td><td>08-916-5D</td><td>Used for dissection (steps 1.3, 3, and 5)</td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td>For cryosectioning (step 7)</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura</td><td>4583</td><td>Used as embedding media (step 6)</td></tr></tbody></table>

obtaining high-quality cryosections of whole rabbit eye

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.

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 <strong class="xf...

Read this Scientific Article Protocol about Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models at JoVE.com

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

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.

Obtaining High-Quality Cryosections of Whole Rabbit Eye

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

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1.5K Views.  University of Southern California. 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. 

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

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Developmental Biology

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Medicine

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Neuroscience

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

Mouse Dorsal Root Ganglion Cryosectioning

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of inflammatory and neuropathic pain, itch, as well as other peripheral neurological conditions. However, it remains a challenge to consistently obtain high-quality, intact, and flat cryostat sections onto glass slides because of the tiny sample size of the DRG tissue. So far, there is no article describing an optimal protocol for DRG cryosectioning. This protocol presents a step-by-step method to resolve the frequently encountered difficulties associated with DRG cryosectioning. The presented article explains how to remove the surrounding liquid from the DRG tissue samples, place the DRG sections on the slide facing the same orientation, and flatten the sections on the glass slide without curving up. Although this protocol has been developed for cryosectioning the DRG samples, it can be applied for the cryosectioning of many other tissues with a small sample size.

Author Spotlight: A Procedure for Mouse Dorsal Root Ganglion Cryosectioning  

Presented here is the development for consistently acquiring high-quality dorsal root ganglion cryostat sections.

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...