Name: Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium (Video) | JoVE Journal
Uploaded: 2011-02-27T16:00:00.000+00:00
Duration: 16 min 47 s
Description: Learn to dissect a mouse eye for whole mount retinal pigment epithelium preparation. This technique enables researchers to study retinal development and diseases using precise microdissection methods.

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This work was supported by the National Institute of Environmental Health Sciences [K22ES012264 to A.J.R.B.] and an American Cancer Society InstitutionalResearch Grant [ACS-IRG-00-173-04]pilot projectaward [to A.J.R.B.]. We also thank members of the Bishop lab for critical reading of the manuscript and comments on the video and Adam Brown in particular, for the example of what not to do. We thank Dr. Donald McEwen of Greehey Children's Cancer Research Institute for allowing us the use of his dissecting scope/ video camera set-up for filming of the dissection video. We thank Daron Brown at Corte Instruments for sharpening and repair of our microdissection tools.

1. Remove Extraneous tissue from the outside of the eye
<ol>
	<li>Pour 1X PBS into the lid of a 35mm dish. The level should be just below the lip of the lid.</li>
	<li>Using straight forceps, transfer eye(s) from the storage tube to the 35mm dish. Eyes are submerged in PBS for two reasons: 1. The extraneous tissues will "float" away from the eye so you can see and remove them easily, and 2. During dissection, the eye will naturally maintain its spherical shape while in suspension, allowing you to work with this shape rather than against it.</li>
	<li>Using both pairs of angled forceps (45° and 15°), gently remove as much muscle and connective tissue as possible, pulling the tissue against the grain (i.e. toward the cornea). Some connective tissue will remain. Effort should be made to squeeze the eyeball as little as possible.</li>
	<li>Trade your 15° forceps for spring scissors. While holding the eye steady with 45° forceps, use the spring scissors to cut away remaining muscle and connective tissue, as well as the optic nerve. *Tip: Use the outside edge of the scissors to push the tissue against the grain (toward the corneo-scleral divide) and then trim. Continue until all extraneous tissue has been removed from the eye.</li>
</ol>
2. Remove the cornea and lens
<ol>
	<li>With the cornea facing up, use the 45° forceps to pinch up a small fold in the center of the cornea.</li>
	<li>Using the spring scissors, make a small incision at the base of the flap. Let go of the flap.</li>
	<li>Holding the eyes still with your forceps, insert the lower scissor blade into and perpendicular to the incision and make a cut toward the corneo-scleral divide. Try to keep your lower blade under the iris so both the cornea and iris are cut. Do not cut all the way to the corneo-scleral divide. Keep scissors inserted in the eye.</li>
	<li>Using your 45° forceps, carefully rotate the eye so that your scissors are parallel to the corneo-scleral divide and make a small cut. Slowly rotate the eye 360°, making small incisions all the way around. Again, try to keep the lower blade of your spring scissors under the iris so both the iris and cornea are cut. Lift the iris and cornea away from the rest of the eye and discard them.</li>
	<li>Tilt the eye on its side and press down gently on the posterior half of the eye, forcing the lens out. Discard the lens. The inside of the remaining eyecup is lined by the neural retina, which should look smooth and opaque.</li>
</ol>
3. Quarter the resulting eyecup, resulting in a 4-"petaled", flower-like structure
<ol>
	<li>The eye should be positioned such that the opening of the eyecup is perpendicular to your body. Use your 45° forceps to steady and position the eyecup.</li>
	<li>Holding your spring scissors in front of you so they are perpendicular to your body, insert the lower blade of your spring scissors into the top edge of the open eye cup. Using your 45° forceps, carefully align the eye cup so that a single, straight cut can be made from the corneo-scleral divide toward the optic nerve head. All cuts should be perpendicular to the corneo-scleral divide and unless specified, should go as close to the optic nerve head as possible without cutting into it.</li>
	<li>Rotate the eyecup 180°, maintaining its perpendicularity with your body. Use technique in step 3.2 to make a second perpendicular cut.</li>
	<li>Rotate the eyecup and repeat the technique described in 3.2 to make two more cuts. All four cuts should be as close to 90° apart as possible. Keep in mind that the eye is not perfectly spherical so there will be some variability.</li>
</ol>
4. Cut each of the four "petals" in half, resulting in an eight-petal flower-like structure
<ol>
	<li>Transfer the quartered eyecup from the 35mm dish to a clean slide. With the eyecup facing up, scoop under it with the 45° forceps. Carefully lift it out of the liquid and place it on a clean slide.</li>
	<li>Using both pairs of angled forceps, gently open out the eye, with the neural retina facing up. DO NOT REMOVE THE NEURAL RETINA- it helps the eye maintain some of its spherical shape so you are not working against it. It also protects the RPE from being accidently damaged in the following procedures.</li>
	<li>Rotate the slide until one of the petals is perpendicular to your body.</li>
	<li>Use your 15° forceps, gently grab the corner of the petal. The corner of the petal should be cornea, not neural retina or RPE. Lift up on the corner gently, allowing it to retain its curvature.</li>
	<li>Insert the bottom blade of your spring scissors between the petal and the slide. Line up your scissors such that a single, straight, perpendicular cut can be made from the corneo-scleral divide toward the optic nerve head. Cut as close to the optic nerve head as possible without cutting into it. Let go of the petal, allowing it to lie on the slide.</li>
	<li>Rotate the slide 90°, so that the next petal is perpendicular to your body. Repeat step 4.5.</li>
	<li>Repeat steps 4.5 and 4.6 twice more. The resulting structure should look like an eight-petal flower.</li>
</ol>
5. Remove the neural retina
<ol>
	<li>Pipette 100-200 μL of 1X PBS onto the specimen. This will prevent the neural retina from sticking as you peel it off of the RPE.</li>
	<li>Typically the neural retina is loosely attached to the RPE. However, it does take practice and skill to remove only the neural retina and not the RPE. Gently grasp a petal of the neural retina with one pair of angled forceps while stabilizing (holding down) the rest of the eye with the other pair of angled forceps. Pull the neural retina away from the rest of the eye. Continue this procedure until the entire neural retina has been removed. *Tip: stick the tip of your 15° forceps into the optic nerve head to anchor the eye. Then use the 45° forceps to sweep up underneath the neural retina and lift it off and away. It is usually best to lift the neural retina from the optic nerve head toward the corneo-scleral divide. Be sure to pull away any loose remnants of the iris. The iris is quite sticky and can otherwise get stuck to the RPE.</li>
	<li>Rinse the specimen with 1X PBS. Pipette 100-200 μL of 1X PBS into the specimen and suck it back up. Discard the PBS. Repeat as necessary to remove dust and debris.</li>
</ol>
6. Mount you specimen(s) on a slide
<ol>
	<li>Pipette 80-90 μL of 90% glycerol onto a new slide in a rectangular shape. Use the side of the pipette tip to make an even, rectangular smear of glycerol. (This is the procedure for mounting two RPE on one slide.)</li>
	<li>Insert the lower arm of 15° forceps under the specimen. Gently lift the specimen off the dissecting slide.</li>
	<li>Slowly lower the specimen into the glycerol. Gently wiggle the forceps as you pull them out from under the specimen.</li>
	<li>Repeat steps 6.1-6.3 for the other specimen.</li>
	<li>Rotate the slide so that the long side is perpendicular to your body. Hold your cover glass at a 45° angle to the slide so that it is touching the slide. Move it toward the glycerol smear until they touch. Maintaining contact between the cover glass and slide, slowly move the cover glass away from the smear, toward the edge of the slide. Line up the long sides of the slide and the cover glass so that they are parallel. Hold the corners of the cover glass between your thumb and forefinger.</li>
	<li>Pick up the 15° forceps in your other hand. Cradle the upper (free) edge of the cover glass in the crook of the arm. Very slowly, lower the cover glass. When contact is made between the cover glass and the glycerol, stop and wait for the glycerol to migrate. Repeat this lowering and waiting until the RPE whole mounts are fully covered. This is to prevent the highly viscous glycerol from capturing air bubbles. DO NOT PUSH DOWN ON THE COVERGLASS - the glycerol will squeeze out from under it, making adhesion very poor.</li>
	<li>With clear nail polish, paint around the perimeter where the cover glass and slide meet.</li>
	<li>Place the slide on top of an empty rack to dry.</li>
</ol>
7. Representative Results:
The result of this procedure should be a structure that looks like a flower and should be fairly symmetrical.
<img src="/files/ftp_upload/2563/2563fig1.jpg" alt="Spintronics schematic: magnetic domain visualization, indicating directional magnetization in diagram." data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. Whole mount RPE from a wildtype C67Bl/6J mouse. RPE from black or agouti animals should be dark brown in color and should have a smooth surface. It is normal to notice undulations in the topography of the petals. The pigmentation on any give specimen may be somewhat variable, due to variable density of pigmentation of both the RPE and the underlying choroid. White arrows point to hypopigmented "channels"- this is normal and is due to the underlying vasculature of the eye. Blue arrow points to physical damage to both the RPE and the underlying choroid.
<img src="/files/ftp_upload/2563/2563fig2.jpg" alt="Thin layer chromatography result; radial separation pattern on silica plate showing compound distribution." data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. Whole mount RPE from a dilute mouse. Specimens harvested from dilute animals can range in color from nearly transparent to café au lait and any one specimen is like to have variability within it, indicated in black circles here. In general, RPE harvested from younger animals are lighter and those harvested from older animals are darker. Black arrows indicate some of the underlying vasculature, which appears hypopigmented.
<img src="/files/ftp_upload/2563/2563fig3.jpg" alt="Fluorescence microscopy images; cellular structure comparison at 50μm scale; research analysis." data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. Phalloidin staining can be used to detect physical damage to the RPE. Phalloidin staining clearly outlines the cell membranes, showing the hexagonal shape of the epithelium. (A) Example from a black mouse. (B) Example from a dilute mouse.
<img src="/files/ftp_upload/2563/2563fig4.jpg" alt="Microscopic image comparison of plant structure diagram detailing cellular arrangement and morphology." data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4. A poorly dissected RPE from a dilute mouse. (A) Brackets indicate margins of cornea that are too wide, which can cause buckling and/ or folding. Within the black circle you can see excessive amounts of extraneous tissue that have not been removed from the back of the eye. They are particularly obvious because the sample is from a dilute animal. The petal on the bottom left is partially folded over. (B) This whole mount has the appearance of a pinwheel. Black arrows indicate some of the cuts that were made. Many of the cuts from the corneo-scleral divide toward the optic nerve head are not in line with the diameter. (C) Black arrows indicate how the cuts should have been made, in line with the diameter and perpendicular to the tangent.

<ol><li>Bishop, A. J., Kosaras, B., Sidman, R. L., Scheistl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Benzo%28a%29pyrene+andX-rays+induce+reversions+of+the+pink-eyed+unstable+mutation+in+the+retinal+pigmentepithelium+of+mice%5BTitle%5D)+AND+%22Mutat.+Res.%22%5BJournal%5D)">Benzo(a)pyrene andX-rays induce reversions of the pink-eyed unstable mutation in the retinal pigmentepithelium of mice.</a> Mutat. Res.. 457, 31-31 (2000).</li>
<li>Reliene, R. H. lavacove, Mahadevan, A., Baird, B., M, W., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diesel+exhaust+particles+cause+increased+levels+of+DNA+deletions+after+transplacental+exposure+in+mice%5BTitle%5D)+AND+%22Mutat.+Res%22%5BJournal%5D)">Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice.</a> Mutat. Res. 570, 245-2452 (2005).</li>
<li>Bishop, A. J., Barlow, C., Wynshaw-Boris, A. J., Scheistl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atm+deficiency+causes+increased+frequency+of+intrachromosomal+homologous+recombination+in+mice%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Atm deficiency causes increased frequency of intrachromosomal homologous recombination in mice.</a> Cancer Res. 60, 395-399 (2000).</li>
<li>Brown, A. D., Claybon, A. B., Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+WRN+helicase+domain+is+not+required+for+spontaneous+homologous+recombination-mediated+DNA+deletion%5BTitle%5D)+AND+%22J.+Nucleic+Acids%22%5BJournal%5D)">Mouse WRN helicase domain is not required for spontaneous homologous recombination-mediated DNA deletion.</a> J. Nucleic Acids. , (2010).</li>
<li>Claybon, A., Karia, B., Bruce, C., Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PARP1+suppresses+homologous+recombination+events+in+mice+in+vivo%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">PARP1 suppresses homologous recombination events in mice in vivo.</a> Nucleic Acids Res. , Forthcoming (2010).</li>
<li>Bishop, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((p53-%2C+and+Gadd45a-deficient+mice+show+an+increased+frequency+ofhomologous+recombination+at+different+stages+during+development%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">p53-, and Gadd45a-deficient mice show an increased frequency ofhomologous recombination at different stages during development.</a> Cancer Res. 63, 5335-5343 (2003).</li>
<li>Bodenstein, L., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+development+of+the+mouse+retinalpigment+epithelium.+Part+I.+Cell+and+tissue+morphometrics+and+topography+of+mitoticactivity%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Growth and development of the mouse retinalpigment epithelium. Part I. Cell and tissue morphometrics and topography of mitoticactivity.</a> Dev Biol. 121, 192-204 (1987).</li>
<li>Burke, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epithelial+phenotype+and+the+RPE%3A+is+the+answer+blowing+in+the+Wnt%3F%5BTitle%5D)+AND+%22Prog+Retin.+Eye+Res%22%5BJournal%5D)">Epithelial phenotype and the RPE: is the answer blowing in the Wnt?</a> Prog Retin. Eye Res. 27, 579-595 (2008).</li>
</ol>

No conflicts of interest declared.

The RPE is the site of the pun assay, an in vivo assay of homology-directed repair. The pun assay has been used to study the effects of different DNA damages1,2 and DNA damage signaling and repair genes3,4,5 on the frequency of homology-directed repair. This assay is highly sensitive, detecting single-cell events on the RPE1 . It can also detect differences in the timing of homology-directed repair events during development6. The...

<table><tbody><tr><td>straight forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-4903</td><td>tip: .08 x .04 mm material: INOX</td></tr><tr><td>45&amp;#x00B0; forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-5005</td><td>tip: .05 x.01 mm material: INOX</td></tr><tr><td>15&amp;#x00B0; &quot;up&quot; forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-5045A</td><td>tip: .1 x.06 mm material: INOX</td></tr><tr><td>spring scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS-5604</td><td>comb. tip width 0.3mm cutting edge length 3mm material: stainless steel</td></tr><tr><td>binocular dissecting microscope</td><td>Carl Zeiss, Inc.</td><td>Discovery V.8</td><td>use reflected light source</td></tr><tr><td>phalloidin</td><td>Invitrogen</td><td>A22283</td><td>Alexa Fluor 546</td></tr></tbody></table>

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

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 ...

My name is Allison Clavon. I'm a research assistant for Dr.Alex Bishop at the Gree Children's Cancer Research Institute at UT Health Science Center in San Antonio, Texas. Today I'm going to demonstrate how to dissect a rodent eye for a whole amount of the retinal pigment epithelium.This tissue is of interest for studying retinal development and diseases and is also the site of an in vivo homology directed DNA repair assay. So let's get started. Here is an eye ready for dissection.First, let's take a look at the anatomical structures you'll use as landmarks throughout the procedure. This eyes from a dilute animal. Here is the optic nerve at the back of the eye opposite.That is the cornea. The iris shows through the cornea. The whitish area at the back of the eye is the sclera.Therefore, this region is called the cornea scleral divide. The first step of dissection is to remove extraneous tissues such as muscle connective tissue and the optic nerve. Next, the cornea must be removed, making a circular cut.At this point, the lens can be popped out resulting in a hollow eye cup. Next four cuts are made from the corneal scleral divide toward the optic nerve head. The resulting structure resembles a four pedaled flower.Each of these four petals is then cut down the middle, resulting in a structure that resembles an eight pedaled flower. At this stage, the I may buckle in areas due to its spherical nature. To combat this and achieve a flatter hole mount, you can make eight shorter cuts just passing the buckle.You'll need several materials handy at your workstation, including a 100 or 200 p pipette and corresponding tips, one X-P-B-S-A 35 millimeter dish slides and cover glass, 90%glycerol and clear nail polish for slide preparation, a rack for drying slides and a box of Kim wipes and a waist disposal container. In addition to these common materials, you'll also need a set of specialized microdissection tools, including straight forceps, 45 degree angled forceps are used to hold, turn, and generally manipulate the eye throughout dissection. These 15 degree forceps have an angle up and are also used for handling and manipulation of the eye.Finally, you'll need a pair of spring scissors. It's very important that you use spring scissors as opposed to traditional style scissors. For this dissection, before you begin dissection, make sure your chair height is adjusted so that when you're sitting up straight, your eyes are level with the oculars.Slouching at the scope causes shoulder and back pain and inhibits the quality of your dissections. To begin pour one XPBS into the lid of the 35 millimeter dish. Then transfer the eyes you plan to dissect into the dish.This is an eye from a wild type C 57 black mouse. You can see it as very small about three and a half millimeters in diameter. The opacity of the sclera gives it a bluish hue.The first step in dissection is to remove all extraneous tissues from around the eye, such as muscle connective tissue and the optic nerve. Here I have the 45 degree forceps in my left hand and the 15 degree forceps in my right hand. You can use one pair of forceps to steady the eye while pulling tissue away with the other.When a large piece of tissue comes free, carefully wipe your Forceps on a Kim wipe. Just be careful not to crush or puncture The eye. One of the benefits of performing this dissection in suspension is that you can easily see the connective tissue floating away from the eye.Eventually, you'll reach a point where you can no longer remove any more tissue using forceps. Trade out your 15 degree angled forceps for your spring scissors. Use the spring scissors to clip off the optic nerve.Use the outside edge of the blade to Push the tissue forward toward the cornea scleral divide. Then trim and gently wipe your scissors on a kim wipe. Let's look at that again.Push the tissue forward toward the cornea scleral divide with the outside edge of your scissors when the tissue Is sticking up. Trim, continue pushing Bits of tissue forward with the blunt edge of your scissors and then trimming them away until all extraneous tissue is removed. There are usually two bands of tough tissue on the eye.The first is attached at the corneal scleral divide, and the second shown being trimmed. Here is about halfway between the corneal scleral divide and the optic nerve head. The next step in dissection is to remove the cornea and lens.Use your 45 degree forceps to pinch up a flap at approximately the middle of the cornea. Then use your spring scissors to make a small incision at the base of the flap. Insert the lower blade of your scissors into the incision under the iris.Make a cut toward, but not into the Corneal scleral divide.Now. Slowly rotate the eye 360 degrees, making small cuts all the way around. Try to keep the scissors under the iris, cutting both the cornea and the iris at the same time.It takes a lot of practice to do it as easily As I show it here. Once the cornea is Removed, turn the eye on its side and gently press on the outside of the eye pushing the lens out. The remaining structure will be cup like next.The eye must be quartered. To do this, turn the eye on its side and insert the lower blade of your spring scissors. Make a longitudinal cut toward, but not all the way to the optic nerve head.To do this, keep your arms at your side and your scissors in front of you approximately perpendicular to your body and the eye cut. Make your second cut directly opposite the first cut. Take your time lining up so that you can make a smooth cut.Making multiple cuts can cause jaggedness, but if Lined up carefully are not noticeable, notice how I'm able to Work with this spherical shape of the eye rather than against it because the eye retains its normal shape. When Suspended in PBS, the rest of the dissection will take place on A slide. Cradle the eye with your 45 degree angled forceps and lift it out of the PBS.Place the eye on a fresh slide. Using both pairs of angled forceps, carefully unfold the eye. Notice there is a small margin of cornea and iris around the perimeter of the eye, which is useful for handling.Next, you need to cut each of the four pedals in half. Rotate the eye so that one of the pedals is perpendicular to your body. Grasp the corner of the pedal with your 15 degree angled forceps.Lift up slightly and then make a cut from the cornea scleral divide toward the optic nerve head. These cuts should be perpendicular to a tangent. A tangent is a line that is perpendicular to the diameter.Therefore, your cuts should be in line with the diameter of the eye. Now rotate the slide so the next pedal is perpendicular to your body and repeat. Grasp the corner of the pedal.Lift up slightly and cut. Repeat until each of the four large pedals has been cut in half. The whitish substance is the neural retina, which directly overlays the RPE.The neural retina can protect the RPE, so leave it intact until all of your cuts have been made. Notice how the pedals are slightly curved. Do not fight against this.Take your time lining up so you can make a good single cut. Because the eye is round, it has a tendency to buckle to prevent this buckling. You can make eight shortcuts.One on each pedal. Use the same basic strategy as the previous round of four cuts. Lift the corner of the pedal and cut, but in this case, only cut About halfway down the length of the pedal.Once all your cuts are made, you Can remove the neural retina. Here I'm adding a few drops of one XPBS to the specimen. You don't wanna let your RPE dry out.The neural retina should only be loosely associated with the underlying RPE. You can see it lifts off quite easily. However, it will usually remain attached at the corneal scleral divide, which on the inside of the eye is a bumpy ridge called the ciliary body.You can insert the tip of your 15 degree forceps into the optic nerve head to anchor the specimen while pulling off the neural retina. The ciliary body marks the edge of the RPE, the darkly pigmented tissue that came off. Here is iris, not RPE.I recommend removing as much of the iris as possible because it is very sticky and can fold over and stick to the RPE if the neural retina is flaky or if remnants of it remains stuck to the RPE. One of two things probably went wrong. Either the eyes were not fixed properly, such as for too long or too short a period of time, or they were in storage for too long before dissection.The pedal I'm uncovering here has not yet had its shortcut. You can see how the pedal about a third of the way from the edge of the RPE. This is why The eight shortcuts are recommended.Once all of the neural Retina has been discarded, use a pipette to rinse the RPE several times with 100 to 200 microliters of PBS until all dust and debris has been removed. Pipette 80 to 90 microliters of 90%glycerol onto a fresh slide in a rectangular shape. Note, this is the optimal volume of glycerol for a 22 by 50 millimeter cover glass and should be adjusted accordingly.For other sizes of cover glass, use your pipette tip to spread the glycerol. Be careful not to add air bubbles to the glycerol. If you do get a bubble, drag it to the edge where it will pop.Slide the lower arm of your 15 degree forceps under the RPE and lift up carefully, slowly lower the RPE into the glycerol. The RPE will float on top of the glycerol and may curl or twist a bit. Use your forceps to straighten the RPE and to drag the glycerol over the surface of the RPE.If any air bubbles form. As you do this, drag them to The edge where they will pop holding the cover glass at about 45 Degrees. Touch it to the edge of the glycerol.Then move it toward the edge of the slide. Hold the bottom of the cover glass with your thumb and forefinger and cradle the opposite edge with your 15 degree forceps. Very slowly lower the cover glass toward the slide as soon as contact is made.With the RPE, stop lowering, allowing the glycerol to migrate continue to lower your cover glass slowly. If you lower the cover glass too fast, air bubbles will become incorporated into your slide here. I will lower the cover glass slowly as it touches the RPE and allow the glycerol to migrate.You can see when I let the cover glass go quickly, air bubbles become incorporated. Use a generous amount of clear nail polish to attach the edge of the cover. Glass to the slide go all the way around the perimeter.This is a typical RPE from a C 57 black mouse. It is normal to see underlying vasculature as indicated here. It is also normal to see some variation in pigmentation such as patches that appear to be less pigmented.However, clear spots such as this are likely to be due to physical damage to the RPE and the underlying choroid. Because the choroid of the eye is also pigmented. It is possible for damage to a black RPE to be unnoticeable if the choroid is left intact.This is a typical RPE from a dilute mouse. As indicated, you will be able to see vasculature and there will be some variation in pigmentation such as darker patches and lighter patches. However, it is nearly impossible to see damage on a dilute RPE.Are these small patches damage or are they normal variation in pigmentation? I suggest foid and staining to address this question. It will give you an idea if you're handling the I two roughly.The stain clearly outlines the cell membranes, making damaged spots obvious, but in all reality, the RPE is fairly resilient, and if you handle the eye by the cornea, you are unlikely to cause damage. Here is a whole mount with a host of problems, the least of which indicated here is too wide a margin of cornea, which can lead to buckling and folding the squiggly material. You can see here is a massive extraneous tissue remaining on the back of the eye.This pedal is partially folded over, and here you can see the RPE has been partially torn. Another problem is that this eye has the appearance of a pinwheel. Here I have drawn in the angles of the cuts.However, this is how they should have been. Remember, your cuts should be perpendicular to the tangent and in line with the diameter. I've just demonstrated how to dissect a mouse eye for a whole mount of the retinal pigment.Epithelium, the most important thing is to maintain good posture, but try to stay relaxed. I hope you found this video useful and wish you all the best with your future experiments.

dissection-mouse-eye-for-whole-mount-retinal-pigment

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

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

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 Mouse Retinal Cryo-sections for Immunohistochemistry

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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.

Microdissection of the Rodent Eye

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

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.

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

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

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.

Cryosectioning and Immunostaining of Mouse Retina

The retina exhibits complex spatial organization across its laminar structure. Modern spatial transcriptomics and sequencing techniques require tissue sections &lt; 20 µm thick, limiting their retinal research applicability. This fresh-frozen cryosectioning method produces thin en face sections of mouse retina while preserving planar spatial relationships, enabling comprehensive molecular mapping across laminae.

En face Cryosectioning of Mouse Retina for High-dimensional Spatial Molecular Analysis

Learn to dissect a mouse eye for whole mount retinal pigment epithelium preparation. This technique enables researchers to study retinal development and diseases using precise microdissection methods.

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

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