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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.
  2. Enucleate the eyes with curved forceps and transfer them to a small petri dish with 0.1 M PBS (Table 1). Under a dissection ....

Representative Results

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.......

Discussion

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.......

Disclosures

The authors declare no competing financial interests.

Acknowledgements

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.).

....

Materials

NameCompanyCatalog NumberComments
16% ParaformaldehydeElectron Microscopy Sciences15710Fixative
AcrylamideFisher BiotechBP170Hydrogel monomer
Axio Imager.Z2ZeissFluorscence microscope
BSAFisher ScientificBP1600Blocking agent
Eclipse TiNikon InstrumentsScanning confocal microscope
KClVWRBDH0258Buffer component
KH2PO4SigmaP5655Buffer component
Na2HPO4Sigma AldrichS9763Buffer component
NaClSigma AldrichS7653Buffer component
NaH2PO4Sigma AldrichS0751Buffer component
NaN3Sigma AldrichS2002Bacteriostatic preservative
NDSAurion900.122Blocking agent
NIS Elements ARNikonImage analysis software
SDSBioRad1610301Delipidation agent
SorbitolSigma Aldrich51876Buffer component
Triton-X-100SigmaT8787Surfactant
Tween-20Fisher ScientificBP337Surfactant
VA-044Wako Chemicals011-19365Thermal initiator

References

  1. Witkovsky, P. Dopamine and retinal function. Documenta Ophthalmologica. 108 (1), 17-40 (2004).
  2. McMahon, D. G., Iuvone, P. M. Circadian organization of the mammalian retina: from gene regulation to physiology and diseases. <....

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