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R. D. Young, B. P. Palka, C. E. Tucker, K. M. Meek, A. J. Quantock; Characterization of Cuprolinic Blue-Contrasted Proteoglycan Structures by Cryoprocessing in Hydrated Cornea of Embryonic Chick. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4535.
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Corneal proteoglycans (PGs) are diffuse hydrated biopolymers consisting of a protein core and 1-5 glycosaminoglycan (GAG) sidechains and appear by electron microscopy as filaments, regularly associated with collagen fibrils, when complexed with cationic dyes such as cuprolinic blue (CB) (Scott & Haigh, Biosci Rep 1985;5:765). The precise relationship between CB-stained filaments and PG structure in native hydrated tissue remains unknown. We applied cryoprocessing methods to embryonic chick cornea +/- CB aiming to preserve native corneal ultrastructure and further characterize PG-collagen organisation during development.
Corneas were obtained from chick embryos at 11-18 days of incubation and processed by i) conventional fixation in glutaraldehyde with CB, ethanol dehydration and epoxy resin embedding; ii) cryofixation by high pressure freezing in a Leica EMPACT2 and freeze-substitution in either: a) 2% osmium-acetone at -90°C and epoxy resin embedding at 20°C, or b) methanol plus CB at -90°C and embedding as before, or in Lowicryl HM20 resin at -50°C. Sections were examined in a Jeol 1010 electron microscope and images obtained of collagen interfibrillar structures using a Gatan Orius CCD camera.
Conventional CB processing revealed PGs as dense collagen-associated filaments at all developmental stages, plus large chondroitinase ABC-sensitive structures up to day 14. Following high pressure freezing and freeze-substitution with CB, or with osmium, PGs were detectable as diffuse filamentous structures. Presumably these were uncollapsed GAG chains, extending into the interfibrillar space. Regular focal densities tightly-apposed to collagen fibrils, were also observed and may represent sites of PG core proteins. Intricate assemblies of fibril-associated PG structures became more condensed through development.
Cryoprocessing by high pressure freezing and freeze-substitution preserves near-native hydrated structure and avoids extensive collapse of hydrated GAG chains, thus revealing more detailed ultrastructure of PGs in developing chick cornea. However PG-collagen associations conform to those found previously by conventional CB processing methods.
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