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Protocol

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Neuroscience

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

Published: March 6th, 2021

DOI:

10.3791/62178

1Eye Research Institute, Oakland University

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 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 µm (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 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.

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....

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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

  1. Euthanize the mouse with an overdose of CO2, followed by cervical dislocation.

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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.......

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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.......

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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.).

....

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Name Company Catalog Number Comments
16% Paraformaldehyde Electron Microscopy Sciences 15710 Fixative
Acrylamide Fisher Biotech BP170 Hydrogel monomer
Axio Imager.Z2 Zeiss Fluorscence microscope
BSA Fisher Scientific BP1600 Blocking agent
Eclipse Ti Nikon Instruments Scanning confocal microscope
KCl VWR BDH0258 Buffer component
KH2PO4 Sigma P5655 Buffer component
Na2HPO4 Sigma Aldrich S9763 Buffer component
NaCl Sigma Aldrich S7653 Buffer component
NaH2PO4 Sigma Aldrich S0751 Buffer component
NaN3 Sigma Aldrich S2002 Bacteriostatic preservative
NDS Aurion 900.122 Blocking agent
NIS Elements AR Nikon Image analysis software
SDS BioRad 1610301 Delipidation agent
Sorbitol Sigma Aldrich 51876 Buffer component
Triton-X-100 Sigma T8787 Surfactant
Tween-20 Fisher Scientific BP337 Surfactant
VA-044 Wako Chemicals 011-19365 Thermal initiator

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