Name: immunostaining of whole-mount retinas with the clarity tissue clearing method
Uploaded: 2021-03-06T14:00:00
Description: Watch this Scientific Journal Video about Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method at JoVE.com

We would like to thank Bing Ye, Nathan Spix, and Hao Liu for technical support. This work was supported by the National Institute of Health Grants EY022640 (D.-Q.Z.) and Oakland University Provost Undergraduate Student Research Award (E.J.A.).

Mouse care and all experimental procedures were conducted according to the National Institutes of Health guidelines for laboratory animals and were approved by the Institutional Animal Care and Use Committees at Oakland University (protocol no. 18071).
NOTE: Names of the solutions and their compositions are listed in Table 1.
1. Tissue preparation
<ol>
	<li>Euthanize the mouse with an overdose of CO2, followed by cervical dislocation.</...

<ol>
	<li>Witkovsky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+and+retinal+function.">Dopamine and retinal function.</a> Documenta Ophthalmologica. 108 (1), 17-40 (2004).</li><li>McMahon, D. G., Iuvone, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+organization+of+the+mammalian+retina:+from+gene+regulation+to+physiology+and+diseases.">Circadian organization of the mammalian retina: from gene regulation to physiology and diseases.</a> Progress in Retinal and Eye Research. 39, 58-76 (2014).</li><li>Prigge, C. 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Modification of the CLARITY protocol for whole-mount retinas. 
We have simplified the CLARITY protocol to achieve adequate polymerization without the need for a vacuum evacuation or desiccation chamber, as is used in most previous studies7,9,11. The polymerization process is inhibited by oxygen, requiring that the sample be isolated from air during the polymerization step of the protocol. However, rather th...

The vertebrate retina is perhaps the most accessible part of the central nervous system (CNS), and it serves as an excellent model for studying the development, structure, and function of the brain. Five classes of neurons in the retina are distributed in three nuclear layers separated by two plexiform layers. The outer nuclear layer (ONL) consists of classical photoreceptors (rods and cones) that convert light into electrical signals. Electrical signals are processed by neurons in the inner nuclear layer (INL), including bipolar, horizontal, and amacrine cells, and then transmitted to retinal ganglion cells (RGCs) in the ganglion cell layer (GCL). RGCs are the output neurons of the retina, with the axons projecting to the brain to contribute to image-forming and non-image-forming visual function. In addition, three types of glial cells (Muller cells, astroglia, and microglia) provide nutrients to neurons and protect neurons from harmful changes in their extracellular environment.
One specialized subpopulation of amacrine cells produces and releases dopamine, an important neuromodulator in the CNS, reconfiguring retinal neural circuits during light adaptation1,2. Dopaminergic amacrine cells (DACs) have a unique feature of morphological profiles. Their somata are located in the proximal INL with dendrites ramifying in the most distal part of the inner plexiform layer (IPL). Axon-like processes of DACs are unmyelinated, thin and long, sparsely branched, and bear varicosities (the sites of dopamine release). They form a dense plexus with dendrites in the IPL, including ring-like structures around the somata of AII amacrine cells. The axons also run through the INL toward the OPL, forming a centrifugal pathway across the retina3. We have demonstrated that DAC processes express receptors in response to glutamate release from presynaptic neurons, including bipolar cells and intrinsically photosensitive retinal ganglion cells (ipRGCs)4,5,6. However, it is unclear whether glutamate receptors express on the axons, dendrites, or both since they are cut off in vertical retinal sections and cannot be distinguished from each other5,6. Immunostaining needs to be carried out in whole-mount retinas to reveal three-dimensional branching of DACs and the presence of glutamate receptors on subcellular compartments. Although the retina is relatively transparent, the thickness of a mouse whole-mount retina is approximately 200 &#181;m, which limits the penetration of antibodies into the deep tissue as well as reduces light penetration for high-resolution imaging due to tissue light-scattering. To overcome these limitations, we adapted the immunostaining compatible tissue hydrogel delipidation method (CLARITY) developed recently for thick brain sections to whole-mount mouse retinas7.
The CLARITY method was originally developed by the Deisseroth laboratory for immunostaining and imaging of thick brain samples7. It uses a strong detergent, sodium dodecyl sulfate (SDS) and electrophoresis to remove the lipid components (that cause tissue light-scattering), leaving the proteins and nucleic acids in place. The removed lipids are replaced with a transparent scaffold made up of hydrogel monomers such as acrylamide to support the remaining protein structure. The cleared tissue can be labeled via immunohistochemistry and imaged with substantially increased light penetration depth through the tissue (up to several millimeters below the tissue surface). Since then, the CLARITY method has been optimized and simplified by several research groups8,9,10. A modified CLARITY protocol uses a passive clearing technique to avoid the possible tissue damage produced by electrophoresis for clearing the whole-brain and other intact organs11. However, this method has not yet been applied to whole-mount retinas. Here, we adapted the passive CLARITY technique for whole-mount retinas to make them more transparent for immunohistochemistry and imaging. We found that a majority of the retinal proteins tested were preserved during this process for immunohistochemistry. Using the refractive index matching solution, we were able to image retinal neurons across the approximately 200 &#181;m thickness from the ONL to the GCL in whole-mount retinas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Fixative</td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Fisher Biotech</td>
			<td>BP170</td>
			<td>Hydrogel monomer</td>
		</tr>
		<tr>
			<td>Axio Imager.Z2</td>
			<td>Zeiss</td>
			<td></td>
			<td>Fluorscence microscope</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Scientific</td>
			<td>BP1600</td>
			<td>Blocking agent</td>
		</tr>
		<tr>
			<td>Eclipse Ti</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td>Scanning confocal microscope</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>VWR</td>
			<td>BDH0258</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma Aldrich</td>
			<td>S9763</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma Aldrich</td>
			<td>S0751</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma Aldrich</td>
			<td>S2002</td>
			<td>Bacteriostatic preservative</td>
		</tr>
		<tr>
			<td>NDS</td>
			<td>Aurion</td>
			<td>900.122</td>
			<td>Blocking agent</td>
		</tr>
		<tr>
			<td>NIS Elements AR</td>
			<td>Nikon</td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>BioRad</td>
			<td>1610301</td>
			<td>Delipidation agent</td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma Aldrich</td>
			<td>51876</td>
			<td>Buffer component</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Surfactant</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337</td>
			<td>Surfactant</td>
		</tr>
		<tr>
			<td>VA-044</td>
			<td>Wako Chemicals</td>
			<td>011-19365</td>
			<td>Thermal initiator</td>
		</tr>
	</tbody>
</table>

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.

Modified CLARITY-processed retinas are optically transparent tissue. 
To formulate a tissue clearing method that is compatible with immunohistochemical applications in the retina while providing adequate delipidation and retaining the structural integrity of the cellular proteins, we adapted the CLARITY tissue clearing method to whole-mount mouse retinas. We were able to simplify the protocol and modify it for whole-mount retinas (see Protocol). After completing tissue hybridization, clearing, and r...

Watch this Scientific Journal Video about Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method at JoVE.com

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

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 ...

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