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James Tan, Jose Gonzalez, MinHee Ko; In situ 3D Distribution of Filamentous Actin in Mouse Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3553.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the 3-dimensional distribution of filamentous (F-actin) in the mouse trabecular meshwork (TM) and ciliary muscle (CM) by multimodal 2-photon excitation fluorescence (TPEF) imaging.
Enucleated C57BL/6 and BALB/c eyes were fixed, permeabilized and incubated with Alexa-568-conjugated phalloidin to label F-actin. The limbus of the eye faced down on coverglass, allowing imaging by inverted TPEF using a 63X objective. Excitation wavelength of 850nm was used in combination with a narrowband filter (425nm ± 10nm) for collagen-associated second harmonics generation (SHG), and wideband filter (635nm ± 45nm) for red fluorescence. Corresponding frozen sections were labeled with phalloidin and antibodies against classic smooth muscle epitopes (calponin, caldesmon, and α-smooth muscle actin (ASMA)) to identify CM.
Collagen SHG imaging permitted localization of Schlemm’s,canal (SC) with reference to sclera in situ. With deeper scanning through SC, a structure was encountered with phalloidin labeling, consistent with TM or SC inner wall endothelium. Scanning through this structure showed phalloidin-associated fluorescence in an apparent cortical distribution in cells for 10-15um before intensely phalloidin-labeled finger-like projections were encountered. These projections looked like muscle fibers, appeared to intermingle with labeled cells in the TM, and were oriented perpendicular to SC. Sagittal views of thin frozen sections revealed low intensity phalloidin labeling in the TM adjacent to SC, but phalloidin labeling in the CM was intense. Calponin, caldesmon and ASMA labeling in the TM was sparse and of low intensity, but labeling in the CM was intense and co-localized with phalloidin labeling.
Phalloidin F-actin labeling in the TM and CM of the intact mouse eye could be visualized by TPEF. TPEF and immunohistochemistry indicate an organization of TM sandwiched between SC and CM, wherein CM bundles appear to intermingle or insert into the TM region in mice.
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