Ultrastructural and immunohistochemical studies of microfibril-associated components in the posterior chamber of the eye

Abstract

Connective tissue microfibrils were observed in tissues prepared with methods believed to minimize the loss of tissue components. The eyes of C57BL/6J mice were fixed with glutaraldehyde followed by either freeze substitution, or embedding in glycol methacrylate, a water-miscible embedding medium, after limited or no dehydration. In these preparations, microfibrils were present within sheet-like layers observed in the posterior chamber of the eye. The material enclosing the microfibrils that formed the layer was also preserved, at least partially, by fixation of the tissue with uranyl acetate or potassium permanganate (KMnO₄) as observed in the chick eye. This microfibril-associated material was found to be composed of heparan sulfate proteoglycan (HSPG) as shown by positive immunostaining for HSPG, as well as by identification of 4.5 nm-wide HSPG double tracks as its major constituent. When a considerable amount of this material was lost in KMnO₄-fixed tissues, the remaining portion was preserved in the form of clusters of about 50 nm in width which were periodically adhered along the length of microfibrils. At the center of each cluster, a minute dark particulate structure was present. It was composed of an approximately 10 nm-wide polygonal assembly of 3.5 nm-wide ring-like structures, and was, in unfixed chick eyes, positively immunostained for fibrillin. The periodicity of HSPG clusters, and of fibrillin, along the length of immunostained microfibrils was similar, ranging from 45 nm to 65 nm. These observations indicate that fibrillin is periodically associated at the surface of “classical” microfibrils, and it may mediate the association of large amounts of HSPG to microfibrils.