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Antonio Bergua, Carina Baumgart, Winfried Neuhuber, Bettina Hohberger; Immunofluorescence of intraocular structures combined with the optical clearing method See Deep Brain (SeeDB). Invest. Ophthalmol. Vis. Sci. 2018;59(9):2142. doi: https://doi.org/.
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Optical clearing methods offer the possibility to see into tissues respecting their tridimensional structure. A new water-based clearing technique, See Deep Brain (SeeDB), was successfully applied on neuronal (e.g. brain) and ocular samples in animals and humans. It is of interest, if the SeeDB method can be combined with immunohistochemistry or histochemical stainings in order to get specific anatomical analyses of the marked targets in their ‘natural’ surrounding.
Immunohistochemistry for neuronal nitric oxide synthase (nNOS) and histochemical NADPH-d staining were combined with the clearing method SeeDB in 16 chicken eyes. Stainings with 0.5% diaminobenzidin (DAB) were added. Macroscopic and microscopic photographs were taken. Additionally, transmission electron microscopic analyses of different ocular tissues were performed after clearing with SeeDB.
(1) A clear histochemical NADPH-d reactivity in combination with SeeDB was not possible as a diffuse, blurred staining was observed. (2) Combination of immunohistochemistry for nNOS-DAB with SeeDB yielded a specific staining of the target cells. (3) nNOS-immunohistochemistry and DAB staining have to be done before SeeDB clearing to get best results. (4) Transmission electron microscopy showed that after SeeDB treatment cells and associated structures remained in their approximately primary form and architecture.
The SeeDB clearing method enables visualization of intraocular structures throughout a transparent sclera. The combination of nNOS-immunohistochemistry with SeeDB yielded a specific staining of the target cells. Using this new clearing method applied with classical staining techniques offer the option to analyze intraocular structures in their natural anatomical surrounding. Qualitative and quantitative histological studies as well as clinical-pathological analyses can be performed by this innovative method.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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