June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Immunostaining of whole-mount retinas with the CLARITY tissue clearing method
Author Affiliations & Notes
  • Elizabeth Alessio
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Dao-Qi Zhang
    Eye Research Institute, Oakland University, Rochester, Michigan, United States
  • Footnotes
    Commercial Relationships   Elizabeth Alessio, None; Dao-Qi Zhang, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 5054. doi:
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      Elizabeth Alessio, Dao-Qi Zhang; Immunostaining of whole-mount retinas with the CLARITY tissue clearing method. Invest. Ophthalmol. Vis. Sci. 2020;61(7):5054.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Immunostaining has been routinely performed on thin vertical retinal sections (~12 μm) rather than in whole-mount retinas (~300 μm in thickness) since the latter limits the penetration of antibodies into the deep tissue as well as light penetration for high-resolution imaging. To overcome these limitations, we adapted the immunostaining compatible tissue hydrogel delipidation method (CLARITY) developed recently for brain sections to whole-mount mouse retinas in order to improve the resolution of circuit wiring and subcellular structures of retinal neurons such as dopaminergic amacrine cells (DACs) for immunohistochemistry (IHC).

Methods : Fixed whole-mount mouse retinas were polymerized with an acrylamide monomer to form a nanoporous hydrogel. Retinas were then passively cleared in 10% sodium dodecyl sulfate (SDS) at 40 oC for two days to minimize protein loss and avoid the tissue damage associated with electrophoretic clearing. After tissue clearing, IHC was performed allowing two days for the incubation of primary and then secondary antibodies before mounting the retinas in a refractive index matching solution for imaging.

Results : Twelve primary antibodies were tested with CLARITY-processed whole-mount retinas. Compared to standard IHC, CLARITY improved the quality of staining against tyrosine hydroxylase (TH) for DACs by revealing fine dendritic structures and rings formed by the axon-like process. Immunostaining with an antibody against the AMPA-receptor subunit GluA2 showed distinct puncta in the inner and outer plexiform layers. Triple labeling of these two antibodies with PSD-95 demonstrated the expression of postsynaptic AMPA receptors on dendrites and axon-like processes of DACs. In addition, we found that antibodies for cone photoreceptors, rod bipolar cells, retinal ganglion cells and glial cells all produced clear images of these cells in whole-mount retinas. The only tested antibody found to be incompatible with CLARITY was against phospho-S6 (pS6), a cell activity marker.

Conclusions : CLARITY renders the retina 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 from whole-mount retinas. The expression of GluA2 subunits on DAC axons suggests that DACs receive unusual input from presynaptic neurons via this subcellular compartment.

This is a 2020 ARVO Annual Meeting abstract.

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