Name: dissection of a mouse eye for a whole mount of the retinal pigment epithelium
Uploaded: 2011-02-27T16:00:00
Description: Watch this Scientific Journal Video about Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium at JoVE.com

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 ...

<ol>
	<li>Bishop, A. J., Kosaras, B., Sidman, R. L., Scheistl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzo(a)pyrene+andX-rays+induce+reversions+of+the+pink-eyed+unstable+mutation+in+the+retinal+pigmentepithelium+of+mice.">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. l. a. v. a. c. o. v. e., Mahadevan, A., Baird, B., M, W., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diesel+exhaust+particles+cause+increased+levels+of+DNA+deletions+after+transplacental+exposure+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm+deficiency+causes+increased+frequency+of+intrachromosomal+homologous+recombination+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+WRN+helicase+domain+is+not+required+for+spontaneous+homologous+recombination-mediated+DNA+deletion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP1+suppresses+homologous+recombination+events+in+mice+in+vivo.">PARP1 suppresses homologous recombination events in mice in vivo.</a> Nucleic Acids Res. , (2010).</li><li>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=p53-,+and+Gadd45a-deficient+mice+show+an+increased+frequency+ofhomologous+recombination+at+different+stages+during+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+development+of+the+mouse+retinalpigment+epithelium.+Part+I.+Cell+and+tissue+morphometrics+and+topography+of+mitoticactivity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+phenotype+and+the+RPE:+is+the+answer+blowing+in+the+Wnt?.">Epithelial phenotype and the RPE: is the answer blowing in the Wnt?.</a> Prog Retin. Eye Res. 27, 579-595 (2008).</li></ol>

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...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>straight forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-4903</td>
<td>tip: .08 x .04 mm material: INOX</td>
</tr>
<tr>
<td>45&deg; forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5005</td>
<td>tip: .05 x.01 mm material: INOX</td>
</tr>
<tr>
<td>15&deg; "up" forceps</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-5045A</td>
<td>tip: .1 x.06 mm material: INOX</td>
</tr>
<tr>
<td>spring scissors</td>
<td>&nbsp;</td>
<td>Roboz</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>&nbsp;</td>
<td>Ziess</td>
<td>Discovery V.8</td>
<td>use reflected light source</td>
</tr>
<tr>
<td>phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A22283</td>
<td>Alexa Fluor 546</td>
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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.

Watch this Scientific Journal Video about Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium at JoVE.com

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

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 ...

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

Research

JoVE Journal

Neuroscience

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 &#181;m (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY&#160;can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.

Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...