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Jutaro Nakamura, Yi-Sheng Chang, Gao Chun, Maria M Campos, Robert N Fariss, Tiziana Cogliati, Anand Swaroop; Three-dimensional imaging and reconstruction of GFP-tagged photoreceptors after retinal tissue clarification.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):281.
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© ARVO (1962-2015); The Authors (2016-present)
To visualize GFP-tagged rod photoreceptor cells in retinal flat-mount tissue after clarification using the optical technique reagent ScaleA2 (Hama, Kurokawa et al. 2011).
Rod photoreceptors were tagged by expressing GFP controlled by the promoter of the rod-specific transcription factor NRL (Nrlp-eGFP). Nrlp-eGFP mouse eyes were dissected and fixed in 4% PFA for 4 hours, then incubated in excess volume of Scale2 (4M urea, 10% glycerol, 0.01% Triton X-100, pH 7.7) at 25 degrees Celsius with gentle shaking for three days. Retinal and sclerochoroidal tissues were dissected intact from the globe, flat mounted, and processed for two-photon excitation fluorescent microscopy imaging (Olympus FV1000MPE). Immature photoreceptors were dissociated from postnatal day 5th Nrlp-eGFP mouse retina and injected into the sub-retinal space of wild type and retinal degeneration model mice Nrl-/-, Cep290rd16/rd16;Nrl-/-. One month after transplantation, eyes were collected and processed as above.
Light scattering was reduced dramatically by ScaleA2 tissue clarification method, and the retinal tissue transparency improved so much as to reveal the whole thickness of retinal layers. Nrlp-eGFP fluorescence was fully retained after the clarification method in wild type retina. Integration of Nrlp-eGFP cells, interaction with the host cells, and outer segment formation were observed.
The simple clarification technique using ScaleA2 could be an alternative to conventional flat mount z-stack three-dimensional imaging applied to the evaluation of structural morphologies of host tissue and transplanted donor cells. The ScaleA2 clarification method can be applied to the study of degenerating photoreceptors and can contribute to a better understanding of the fate of transplanted cells in cell-based therapy paradigms.
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