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R. Young, T. O. Akama, M. Liles, M. N. Fukuda, J. R. Ralphs, B. Caterson, A. J. Quantock; Ultrastructural Studies of Developing Chick Cornea Prepared by High Pressure Freezing. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1484.
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© ARVO (1962-2015); The Authors (2016-present)
We utilised high pressure freezing and freeze substitution to attempt preservation of native hydrated tissue ultrastructure in developing chick cornea, avoiding changes known to be induced by conventional aqueous processing methods. Tissue structure and immunolocalization of glycosaminoglycan (GAG) epitopes were compared with specimens processed by chemical fixation.
Corneas were obtained from chick embryos at different stages of development between 8 and 18 days, spanning the time period over which the tissue becomes transparent. They were processed for electron microscopy either by: i) high pressure cryofixation at 2000bar, freeze substitution in acetone:2% osmium at -90°C and epoxy resin embedding, or ii) aqueous fixation in 1% glutaraldehyde, dehydration in ethanol at progressively lowered temperature and Lowicryl resin embedding at -25°C. Immunostaining was carried out on resin sections using antibodies 5D4 and anti "i", reactive with high-sulfated keratan sulfate (KS) and unsulfated polylactosamine respectively, and detected with goat anti-mouse or anti-human, 10nm gold-labelled secondary antibodies.
Good ultrastructural preservation was achieved by high pressure freezing except in the mid stromal region of highly-hydrated, early-stage chick corneas, which showed signs of ice crystal damage. Antibody reactivity was preserved in high pressure frozen samples in spite of the use of osmium tetroxide in the freeze-substitution medium. High-sulfated KS was detected at all developmental stages examined in association with collagen in fibril bundles displaying different fibril orientations. Dense labelling was occasionally found associated with the plasma membranes of keratocytes. In contrast, only low levels of labelling for unsulfated polylactosamine were found in the extracellular matrix, the antibody locating mainly intracellular sites around ER, Golgi and cytoplasmic vesicles.
High pressure freezing offers advantages for improved structural preservation in ultrastructural studies of developing chick cornea. By day 8 of development, matrix KS GAG chains are already substituted with sulfate groups and lack unsulfated domains. Unsulfated polylactosamine, detectable in the intracellular compartment, may represent KS chains prior to sulfation before export from the cell, or other polylactosamine substrates, including lysosomal associated membrane proteins. We postulate that the functional matrix role of KS in corneal stroma requires extensive sulfation of KS chains.
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