Dr. Alaknanda Mishra, Department of Cell Biology and Human Anatomy, University of California Davis, USA, trained us in this method at the National Institute of Immunology, New Delhi. This work was supported by the core grant received from the Department of Biotechnology, Government of India to the National Institute of Immunology, New Delhi. P.S. was granted a research fellowship by the Department of Biotechnology.

This procedure was approved by the Institutional Animal Ethics Committee of the National Institute of Immunology, New Delhi. The serial reference number of the approval is IAEC#480/18. The experiments were performed in accordance with the regulation guidelines of the Committee for Control and Supervision of Experiments on Animals, Ministry of Fisheries, Animal Husbandry and Dairying, Government of India, under the supervision of a professional Veterinarian at the SAF, NII.
1. Preparation
<ol>
	<li>Dilate the rodent eyes using a drop of 0.8% tropicamide and 5% phenylephrine ophthalmic solutions in each eye. Place the animal in a dark area for 30 min before the procedure. 
	NOTE: An analgesic is not required as the animal is euthanized immediately after completing the 30 min. The dark adaptation and dilating the animal&#39;s eyes allow the observer to check for ocular tears and damage before the procedure. Additionally, dilation is advantageous while working with the pigmented eyeball tissue.</li>
	<li>Euthanize the animal by intraperitoneally administering a 5x dose of anesthesia. A typical dose is 200 &#181;L of PBS containing 10 mg of ketamine and 1 mg of xylazine. 
	NOTE: The dose for euthanizing the animal is ketamine at 400-500 mg/kg body weight and xylazine at 50 mg/kg body weight given intraperitoneally. For a typical mouse weighing 20 g, the dose would be 200 &#181;L of PBS containing 10 mg of ketamine (100 &#181;L of a stock solution of 100 mg/mL ketamine) and 1 mg of xylazine (100 &#181;L of a stock solution of 10 mg/mL xylazine).</li>
	<li>Confirm the complete lack of response to a stimulus by examining the pedal reflex. The absence of a heartbeat and respiration are also important indicators.</li>
	<li>Place the mouse in lateral recumbency to allow complete access to the eye.</li>
	<li>Prepare Colibri suturing forceps, curved forceps, a micro-blade (15&#176;, 3 mm in size), spring micro-scissors, and angled spring micro-scissors ready for the procedure.</li>
</ol>
2. Enucleation of the mouse eye along with the third eyelid
<ol>
	<li>Place the mouse in lateral recumbency to allow for complete access to the eye. Open the outer eyelids with the forceps to optimally visualize the third eyelid. 
	NOTE: The third eyelid, also known as the nictitating membrane, is located toward the upper corner of the nasal tangent (Figure 1A).</li>
	<li>Identify the third eyelid as a minor translucent protuberance with a pigmented boundary (Figure 1B).</li>
	<li>Place curved forceps around the eyeball, and pinch to free the eye from extraneous attachments. This will minimize bleeding into the eye from the surrounding tissue.</li>
	<li>Replace the curved forceps with spring micro-scissors, and cut around the eyeball with the third eyelid attached to it. Use small continuous cuts to release the eyeball from the surrounding tissue attachments.</li>
	<li>Place curved spring micro-scissors under the eyeball to free the eye by cutting the optic nerve. Ensure that the eyeball is completely released from the eye socket.</li>
</ol>
3. Clearing the extraneous tissue
<ol>
	<li>Place the enucleated eye from the euthanized mouse in a Petri dish with phosphate-buffered saline (PBS) buffer at pH 7.4, with the Petri dish filled such that the eyeball is submerged in PBS.</li>
	<li>Immobilize the enucleated eyeball of the mouse with the attached third eyelid with one hand using forceps.</li>
	<li>Suspend the eyeball in liquid medium to permit more efficient handling. Some extraocular muscles or tissues will begin to float away from the eyeball. Remove them by cutting them off the eyeball using spring micro-scissors.</li>
	<li>Cut and remove the optic nerve from the base of the eyeball using micro-scissors to allow the better separation of the retina for whole-mount preparations. 
	NOTE: It is not necessary to remove the optic nerve for tissue sectioning.</li>
	<li>Wash the eyeball with PBS, and transfer it into a 1.5 mL tube containing 1 mL of 4% paraformaldehyde (PFA) for a minimum of 2 h at 4 &#176;C for tissue fixation. 
	NOTE: The procedure can be paused at this step by leaving the tissue in PFA for up to 12 h at 4 &#176;C. However, for the isolation of live cells, the fixation steps should not be performed, and the entire procedure needs to be performed without pauses.</li>
</ol>
4. Dissection of the enucleated eye to obtain the anterior and posterior eyecups
<ol>
	<li>After the PFA fixation (if collecting live cells, proceed without the fixation of the eyeball), transfer the eyeball into a Petri dish containing PBS, and place it under a dissecting microscope to perform the subsequent procedure.</li>
	<li>Immobilize the eyeball by holding down the third eyelid firmly with Colibri suturing forceps.</li>
	<li>Make a small incision at the limbus by piercing it using a micro-blade. The limbus is the corneal-scleral junction. Make the incision preferably opposite the third eyelid, thereby making an access point for the spring micro-scissors. 
	NOTE: Using a microblade of 15&#176; and 3 mm in size may provide better control over the depth of this incision.</li>
	<li>Next, using this incision as a starting point, with a pair of micro-scissors, make small and continuous cuts along the circumference of the corneal-scleral limbus. First, complete a semicircle of cuts starting from the incision point to the third eyelid. Then, complete the remaining semicircle of cuts on the other side. Finally, make a cut around the third eyelid to separate the posterior cup and the anterior cup. 
	NOTE: Depending on the eyecup of interest, the nictitating membrane can be left attached to either the anterior or posterior eyecup.</li>
	<li>Remove the lens from the posterior cup using forceps to obtain the posterior cup comprising the neural retina and retinal pigment epithelium (RPE) layers. The lens, posterior cup, and anterior cup of the eye are, thus, separated. 
	NOTE: If working with unfixed tissues, the anterior and posterior cups can now be enzymatically digested to obtain a corneal or retinal single-cell suspension.</li>
	<li>For fixing the tissues, transfer these cups into a 1.5 mL tube containing 1 mL of 4% PFA for 12 h at 4 &#176;C. 
	&#8203;NOTE: Post-fixation, the tissue can be embedded in an appropriate medium, and cryo-sectioning or paraffin sectioning can be done to obtain corneal or retinal sections.</li>
</ol>
5. Isolation of the neural retina and RPE layers
<ol>
	<li>Reintroduce the posterior cup into the Petri dish containing PBS buffer to submerge the tissue.</li>
	<li>Check for the presence of the neural retina. This can be visualized as an opaque layer on the pigmented RPE layer, which is attached at the peripheral circumference of the cup.</li>
	<li>Hold the third eyelid with forceps to immobilize the posterior cup, and peel off the neural retina starting from the periphery using a second pair of forceps. 
	NOTE: Very gentle and slight movements of the hand are highly recommended in order to not damage the tissue. At the optic nerve exit, the curved spring scissors can be inserted under the neural retina, and a cut can be made to completely free the layers if the neural retina remains attached as depicted in Figure 3. Optionally, free or detach the retina with gentle strokes using a soft brush. However, it would not be appropriate to do so if collecting the retina for histology. 
	&#8203;NOTE: The neural retina and pigmented RPE layer with the third eyelid are, thus, obtained.</li>
</ol>
6. Segmentation of retinal and RPE layers for wholemounts
<ol>
	<li>Reintroduce the anterior and posterior cups into the Petri dish containing PBS buffer, and place the dish under the dissecting microscope.</li>
	<li>Immobilize the anterior cup by holding down the third eyelid.</li>
	<li>Use angled spring micro-scissors to make a cut of around 3/4 of the diameter of the eyecup next to the third eyelid, cutting from the periphery perpendicularly toward the optic center.</li>
	<li>Maintain a grip on the third eyelid, and make a cut next to the first cut.</li>
	<li>Make a similar cut between the second cut and the third eyelid. 
	NOTE: Three or four such equidistant cuts open the hemispherical cups into a flower shape, which falls flat and can be easily mounted.</li>
	<li>Immobilize the posterior cup by holding down the third eyelid.</li>
	<li>Follow the same steps as given for making the cuts (steps 6.3-6.5) on the anterior cup to make three or four equidistant cuts on the posterior cup. 
	NOTE: For the histology of the neural retina isolated in step 5, make similar cuts to ensure it falls flat on a surface. Do not make very deep cuts to the center of the cup, as this may make the leaf sections fall apart or separate easily.</li>
	<li>Perform the staining, and whole-mount the tissues for effective visualization7,8. 
	NOTE: The presence of the nictitating membrane acts as a constant physical indicator of the nasal side of the eyecup in wholemounts. It helps to demarcate the dorsal/ventral side of the eyecup, as the third eyelid of the eyecup indicates the nasal/ventral orientation.</li>
</ol>

<ol>
	<li>Choi, 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=Studying+cancer+immunotherapy+using+patient-derived+xenografts+(PDXs)+in+humanized+mice.">Studying cancer immunotherapy using patient-derived xenografts (PDXs) in humanized mice.</a> Experimental and Molecular Medicine. 50 (8), 1-9 (2018).</li><li>Sutherland, C., 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=Laser+Capture+Microdissection+of+Highly+Pure+Trabecular+Meshwork+from+Mouse+Eyes+for+Gene+Expression+Analysis.">Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis.</a> J Vis Exp. (136), e57576 (2018).</li><li>Fernandez-Godino, R., Garland, D. L., Pierce, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+characterization+of+primary+mouse+RPE+cells.">Isolation, culture and characterization of primary mouse RPE cells.</a> Nature Protocols. 11 (7), 1206-1218 (2016).</li><li>Shang, P., Stepicheva, N. A., Hose, S., Zigler, J. S., Sinha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cell+cultures+from+the+mouse+retinal+pigment+epithelium.">Primary cell cultures from the mouse retinal pigment epithelium.</a> Journal of Visualized Experiments. (133), e56997 (2018).</li><li>Claybon, A., Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+mouse+eye+for+a+whole+mount+of+the+retinal+pigment+epithelium.">Dissection of a mouse eye for a whole mount of the retinal pigment epithelium.</a> Journal of Visualized Experiments. (48), e2563 (2011).</li><li>Steven, P., 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=Experimental+induction+and+three-dimensional+two-photon+imaging+of+conjunctiva-associated+lymphoid+tissue.">Experimental induction and three-dimensional two-photon imaging of conjunctiva-associated lymphoid tissue.</a> Investigative Ophthalmology and Visual Science. 49 (4), 1512-1517 (2008).</li><li>Tual-Chalot, S., Allinson, K. R., Fruttiger, M., Arthur, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+immunofluorescent+staining+of+the+neonatal+mouse+retina+to+investigate+angiogenesis+in+vivo.">Whole mount immunofluorescent staining of the neonatal mouse retina to investigate angiogenesis in vivo.</a> Journal of Visualized Experiments. (77), e50546 (2013).</li><li>Ullmann, J. F., Moore, B. A., Temple, S. E., Fernandez-Juricic, E., Collin, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+wholemount+technique:+A+window+to+understanding+the+brain+and+behaviour.">The retinal wholemount technique: A window to understanding the brain and behaviour.</a> Brain, Behavior and Evolution. 79 (1), 26-44 (2012).</li><li>Nakao, S., Hafezi-Moghadam, A., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatics+and+lymphangiogenesis+in+the+eye.">Lymphatics and lymphangiogenesis in the eye.</a> Journal of Ophthalmology. 2012, 783163 (2012).</li><li>Mishra, A., 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=Peripheral+blood-derived+monocytes+show+neuronal+properties+and+integration+in+immune-deficient+rd1+mouse+model+upon+phenotypic+differentiation+and+induction+with+retinal+growth+factors.">Peripheral blood-derived monocytes show neuronal properties and integration in immune-deficient rd1 mouse model upon phenotypic differentiation and induction with retinal growth factors.</a> Stem Cell Research and Therapy. 11 (1), 412 (2020).</li></ol>

The authors have nothing to disclose.

Ocular microdissection has been found to be a difficult task owing to the small size and spherical shape of the rodent eye, and the rodent eye requires innovative techniques for efficient handling8. 
In the current demonstrated method, the enucleated mouse eyeball is obtained with the third eyelid attached for effective and easy handling. Using the third eyelid, the eyeball can be immobilized completely, which allows the dissection to proceed with ease and with minimal ...

Ocular dissection is an important technique in ophthalmic research and has allowed investigators to access the segments of the eye for targeted studies. Previously, ocular researchers relied on the ocular tissue from diseased individuals for their studies. However, the progressively growing number of strains of ophthalmic rodent models1 over the years has diminished the need for human ocular tissue. These mouse strains have permitted a deeper understanding of ocular disease and interventions. Yet, they have also generated a need for innovative techniques of ocular micro-dissection. The small size and limited area of operation severely constrain effective access to the ocular sub-parts. Further, owing to the homogenous cellular assembly of the posterior and anterior eyecups, it is difficult to conduct targeted interventions post-dissection. The current micro-dissection techniques of laser2 and surgical microdissection3,4 are inadequate in meeting such requirements of ocular research. Laser micro-dissection is very effective in single-cell analysis, but the specific tissue needs to be micro-dissected before the laser procedure2. The technique can isolate small regions of interest from a pre-dissected tissue for molecular analysis. Thus, the technique is not suitable for preparing wholemounts or for the isolation of axially packed ocular layers for optimum visualization.
The surgical method is the most widely used technique; this method involves immobilizing the eye via the optic nerve5 and then performing the dissection. This practice is arduous and can damage any fragile tissue, as the spherical eye continues to move during dissection. Despite being beneficial for isolating the various sections of the retinal layers, the technique cannot demarcate the spatial orientation of the tissue upon dissection.
During dissection, maintaining the presence of the attached nictitating membrane or the third eyelid (Figure 1) presents unique and significant advantages. In this method, first, the eyeball is enucleated with the third eyelid. Then, the third eyelid is used to immobilize the eye6 (Figure 2A). This is followed by piercing the eyeball through the corneal limbus and using the incision as the point of entry (Figure 2B,C). Then, the eyecups are separated by cutting along the circumference anteriorly and posteriorly (Figure 2D-G). By dissecting the posterior eyecup further, the translucent layer of the neural retina can be identified and gently peeled off. Three or four equidistant cuts are then made in the obtained hemispherical anterior and posterior cups, which allow these flower-shaped cups to fall flat onto a slide (Figure 2H).
The third eyelid aids in easy and efficient handling during the dissection, thus ensuring minimal damage to the tissue while accessing the various ocular layers and when producing wholemounts. Further, the presence of the third eyelid helps to locate and examine localized interventions during visualization.
The procedure, in our lab, has been performed on a CBA/J or an rd1 mouse strain at P28 of any sex. The procedure can be performed on any strain, age, or sex of animal and has no bias according to these characteristics.
The animals were procured from commercial sources (see Table of Materials) and maintained at the Small Animal Facility (SAF) at the National Institute of Immunology (NII). They were kept in individual ventilated cages (IVC) and received ad libitum access to acidified autoclaved water and food. They were maintained at 21-23 &#176;C and with a 14 h/10 h light/dark cycle.
Given below is a modified surgical method for the micro-dissection of a mouse eye.

<table><tbody><tr><td>Acetaminophen (Biocetamol)</td><td>EG Pharmaceuticals</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Alkaline Phosphatase Kit (DEA)</td><td>Coral Clinical System, India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Automated analyser</td><td>Tulip, Alto Santracruz, India</td><td>Screen Maaster 3000</td><td>Biochemical analyser for liver functional test</td></tr><tr><td>Betadine (Povidon-Iodine Solution)</td><td>Win-Medicare;&amp;nbsp; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Biological safety cabinet ( Class I)</td><td>Kartos international; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Bright Field Microscope</td><td>Olympus, Japan</td><td>LX51</td><td></td></tr><tr><td>CBA/J inbred mice</td><td>The Jackson Laboratory</td><td>Stock No. 000654</td><td></td></tr><tr><td>Cefotaxime (Taxim)</td><td>AlKem ; India</td><td></td><td>cefotaxime sodium injection, No specific Catalog Number (Local Procurement)</td></tr><tr><td>Cell Strainer</td><td>Sigma ; US</td><td>CLS431752</td><td></td></tr><tr><td>Collagenase Type I</td><td>Gibco by Life Technologies</td><td>17100-017</td><td></td></tr><tr><td>Cotton Buds</td><td>Pure Swabs Pvt Ltd ; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>DPX Mountant</td><td>Sigma ; US</td><td>6522</td><td></td></tr><tr><td>Drape Sheet</td><td>JSD Surgicals, Delhi, India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma ; US</td><td>HT110132</td><td></td></tr><tr><td>Forceps</td><td>Major Surgicals; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Gas Anesthesia System</td><td>Ugo Basile; Italy</td><td>211000</td><td></td></tr><tr><td>Glucose</td><td>Himedia, India</td><td>GRM077</td><td></td></tr><tr><td>Hair removing cream (Veet)</td><td>Reckitt Benckiser , India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Hematoxylin Solution, Mayer&#039;s</td><td>Sigma ; US</td><td>MHS16</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Himedia; India</td><td>RM554</td><td></td></tr><tr><td>Hyaluronidase From Sheep Testes</td><td>Sigma ; US</td><td>H6254</td><td></td></tr><tr><td>I.V. Cannula (Plusflon)</td><td>Mediplus, India</td><td>Ref 1732411420</td><td></td></tr><tr><td>Insulin Syringes</td><td>BD ; US</td><td>REF 303060</td><td></td></tr><tr><td>Isoflurane ( Forane)</td><td>Asecia Queenborough</td><td>No B506</td><td>Inhalation Anaesthetic</td></tr><tr><td>Ketamine (Ketamax)</td><td>Troikaa Pharmaceuticals Ltd.</td><td></td><td>Ketamine hydrochloride IP, No specific Catalog Number (Local Procurement)</td></tr><tr><td>Meloxicam (Melonex)</td><td>Intas Pharmaceuticals Ltd; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Micro needle holders straight &amp;amp; curved</td><td>Mercian ;&amp;nbsp; England</td><td>BS-13-8</td><td></td></tr><tr><td>Micro needle holders straight &amp;amp;&lt;br/&gt; curved</td><td>Mercian ;&amp;nbsp; England</td><td>BS-13-8</td><td></td></tr><tr><td>Microtome</td><td>Histo-Line Laboratories, Italy</td><td>MRS3500</td><td></td></tr><tr><td>Nylon Thread</td><td>Mighty ; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Paraformaldehyde</td><td>Himedia; India</td><td>GRM 3660</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Refresh Tears/Eyemist Gel</td><td>Allergan India Private Limited/Sun Pharma, India</td><td>P3060</td><td>No specific Catalog Number</td></tr><tr><td>RPMI</td><td>Himedia; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Scalpel</td><td>Major Surgicals; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Scissors</td><td>Major Surgicals; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>SGOT (ASAT) KIT</td><td>Coral Clinical System, India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>SGPT (ALAT) KIT</td><td>Coral Clinical System, India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Shandon Cryotome E Cryostat</td><td>Thermo Electron Corporation ; US</td><td></td><td>No specific Catalog Number</td></tr><tr><td>Sucrose</td><td>Sigma ; US</td><td>S0389</td><td></td></tr><tr><td>Surgical Blade No. 22</td><td>La Medcare, India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Surgical Board</td><td>Locally made</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Surgical White Tape</td><td>3M India ; India</td><td>1530-1</td><td>Micropore Surgical Tape</td></tr><tr><td>Sutures</td><td>Ethicon, Johnson &amp;amp; Johnson, India</td><td>NW 5047</td><td></td></tr><tr><td>Syringes (1ml, 26 G)</td><td>Dispo Van; India</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>Trimmer (Clipper)</td><td>Philips</td><td>NL9206AD-4 DRACHTEN QT9005</td><td></td></tr><tr><td>Weighing Machine</td><td>Braun</td><td></td><td>No specific Catalog Number (Local Procurement)</td></tr><tr><td>William&#039;s E Media</td><td>Himedia; India</td><td>AT125</td><td></td></tr><tr><td>Xylazine (Xylaxin)</td><td>Indian Immunologicals Limited</td><td></td><td>Sedative, Pre-Anaesthetic, Analgesic and muscle relaxant</td></tr></tbody></table>

microdissection of the rodent eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

A wholemount of rd1 mouse eye/corneal tissue was prepared to study potential lymph-angiogenesis in the anterior/corneal tissue in a diseased state. The attached conjunctival tissue from the third eyelid acted as a positive control, since the cornea lacks lymphatic vessels. For the study, the corneal tissue was dissected with the conjunctiva and was fixed with 4% PFA, followed by permeabilization and blocking. The tissue was then stained with a primary antibody against the lymphatic endothelial marker (LYVE1)<sup class="x...

Watch this Scientific Journal Video about Microdissection of the Rodent Eye at JoVE.com

Microdissection of the Rodent Eye

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

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4.4K Views.  National Institute of Immunology. Oculair onderzoek in vroegere tijden was vooral gebaseerd op het oogweefsel van overleden personen. In de loop der jaren zijn er echter veel oogheelkundige knaagdiermodellen ontwikkeld en is de behoefte aan menselijk oogweefsel afgenomen. Vanwege de kleine omvang en het beperkte operatiegebied in het oog van het knaagdier, is de effectieve toegang tot de oculaire subonderdelen ernstig beperkt.Typisch, om het oog van een knaagdier te opereren, wordt het oog geïmmobiliseerd via de oogze...

Video: Microdissection of the Rodent Eye

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Neuroscience

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Biology

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 ...

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 ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

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 ...

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 ...

Microdissection of the Rodent Eye

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