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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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;&#160; 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&#39;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 &#38; curved</td>
			<td>Mercian ;&#160; England</td>
			<td>BS-13-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holders straight &#38; 
			curved</td>
			<td>Mercian ;&#160; 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 &#38; 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&#39;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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4.1K Views.  National Institute of Immunology. 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. 

Video: Microdissection of the Rodent Eye

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their lifetime. NAION results in optic nerve edema, selective loss of the retinal ganglion cell neurons (RGCs) and atrophy of the optic nerve. A rodent model of NAION that expresses most NAION features and sequelae has been developed, which is applicable to both rats and mice. This model utilizes a focal laser application of 532 nm wavelength to illuminate a photoactive dye, Rose Bengal (RB), to cause capillary damage and leakage at the targeted anterior optic nerve (the laminar region). After rNAION induction, there is an early optic nerve ischemia, optic nerve edema, and intraneural inflammation, followed by selective RGC and axonal loss. Since the optic nerve is a CNS white matter tract, the rNAION model is applicable to mechanistic studies of selective white matter ischemia, as well as neuroprotective analyses and short and long-term mechanisms of glial and neuronal response to ischemia.

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy rNAION

The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo.

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...