Collagen-associated sulphated proteoglycans. Ultrastructure after formaldehyde-cetylpyridinium chloride fixation

Abstract

In the course of an ultrastructural cytochemical study of intracellular sulphated proteoglycans involving the addition of cetylpyridinium chloride in the primary aldehyde fixative, a remarkable ultrastructural preservation of the collagen-associated sulphated proteoglycans was observed. Together with the preservation of their localization among the collagen fibrils (with, for some of them, a 50 nm periodic association with d-bands) and of their native elongated shape, previously observed under similar technical conditions, these stick-shaped and chondroitinase ABC-sensitive proteoglycans exhibited a typical pattern with several dense longitudinal parallel tracks (periodicity: 3-4 nm) not described as yet. Readily observable without high iron diamine-staining, the morphology of these cetylpyridinium chloride-precipitated and collagen-associated polyanions was particularly enhanced after incubation in the diamine solution which ascertained their sulphate content. Such a common ultrastructural organization with parallel tracks for both intracellular (i.e., in eosinophilic polymorphonuclear cells and Kurloff cells) and extracellular CPC-precipitated sulphated proteoglycans could correspond to intrinsic properties of the complexed molecules and could be related to 'double track' proteoglycans observed under other technical conditions in basement membranes.

Kurloff cell ultrastructure after combined formaldehyde-cetylpyridinium chloride fixation and high-iron diamine staining

“…While granular in the absence of CPC, when CPC was added to the primary aldehyde fixative, they showed, as expected, an elongated shape mainly orthogonal to the long axis of the fibrils. Moreover, they exhibited the remarkable longitudinal tracks reported previously by Landemore et al (1991). These longitudinal tracks were readily perceptible in the absence of HID-staining, following the sole post-fixation by ferrocyanide-reduced osmium tetroxide.…”

“…The HID reactivity of the Kurloff cell anionic sites was dependent: on the presence of CPC in the aldehyde fixative in a similar manner to the previously described (Malchiodi Albedi et al, 1988;Landemore et al, 1991) HID reactivity of collagen-associated sulphated proteoglycans.…”

“…The elongated shape of collagen-associated proteoglycans observed on the same sections, was nevertheless well correlated (i) to data from rotary shadowing (Iozzo et al, 1982) and (ii) to CB staining pictures (Scott, 1980;Van Kuppevelt et at., 1984;V61ker et al, 1986). Moreover, the longitudinal tracks of CPC-precipitated collagenassociated proteoglycans (Landemore et al, 1991) presented strong analogies with 'double tracks' of basement membrane proteoglycans previously observed under other technical conditions by Inoue et aI. (1989).…”

“…This was achieved after cetylpyridinium chloride(CPC)-aldehyde fixation. CPC, a quaternary ammonium compound, was added to the fixative solution because (i) it formed an alcohol-and water-insoluble complex with glycosaminoglycans (Scott, 1955), and it effectively provided increased tissue retention of sulphated proteoglycans (Williams & Jackson, 1956;Conklin, 1963;Malchiodi Albedi e~ a]., 1988;Young et al, 1989;Chardin et aI., 1990), and (ii) it was shown to preserve the native elongated shape (Malchiodi Albedi et al, 1988) and to allow visualization of the longitudinal tracks (Landemore et al, 1991) of the collagen-associated sulphated proteoglycans.…”

Preservation of structure and immunoreactivity at the vitreoretinal interface of the rabbit eye

“…Here, several strategies were adopted to preserve structure while retaining antigenicity at the vitreoretinal interface of the rabbit eye. Cetylpyridinium chloride (CPC), a surfactant employed to prevent leaching of proteoglycans and hyaluronan from fixed tissues [25,26], was included in all fixatives. Also, whole eyes, fixed by arterial perfusion, were embedded before subsequent dissection into smaller specimens.…”