Immunolocalization of Collagen Types II and III in Single Fibrils of Human Articular Cartilage

Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmium-acetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several non-collagenous components of the extracellular matrix.

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Immunolocalization of type III collagen in human articular cartilage prepared by high-pressure cryofixation, freeze-substitution, and low-temperature embedding.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.4.7897183 ◽

1995 ◽

Vol 43 (4) ◽

pp. 421-427 ◽

Cited By ~ 17

Author(s):

R D Young ◽

P A Lawrence ◽

V C Duance ◽

T Aigner ◽

P Monaghan

Keyword(s):

High Pressure ◽

Articular Cartilage ◽

Polyclonal Antibodies ◽

Freeze Substitution ◽

Immunogold Electron Microscopy ◽

Type Iii Collagen ◽

Human Articular Cartilage ◽

Chemical Fixation ◽

Osteoarthritic Cartilage ◽

Type Iii

We localized Type III collagen by immunogold electron microscopy in resin sections of intact normal and osteoarthritic human articular cartilage. Comparisons of antibody staining between tissue prepared by high-pressure cryofixation and freeze-substitution without fixatives and that exposed to conventional mild chemical fixation with paraformaldehyde showed that dedicated cryotechniques yielded superior preservation of epitopes that are modified by chemical fixation, and simultaneously provided good ultrastructural preservation. Type III collagen was detected with two polyclonal antibodies, one against the triple-helical domain of the molecule and a second against the more antigenic, globular amino pro-peptide domain, which in this collagen is retained in the extracellular matrix after secretion. Positive labeling was seen in association with the major interstitial fibrils, suggesting co-polymerization of Types III and II collagen in cartilage. Type III collagen could not be detected in aldehyde-fixed normal cartilage. In fixed osteoarthritic cartilage, Type III was detectable only when the antibody to the amino pro-peptide was employed. In contrast, high-pressure cryofixation and freeze-substitution preserved epitopes for both antibodies, permitting immunodetection of Type III collagen in normal and osteoarthritic cartilage. Cryotechniques offer exciting possibilities for significantly improving the immunolocalization of collagens and other fixative-sensitive antigens in situ.

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In Situ Localization of Two Fibrillar Collagens in Two Compact Connective Tissues by Immunoelectron Microscopy After Cryotechnical Processing

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549704500115 ◽

1997 ◽

Vol 45 (1) ◽

pp. 119-128 ◽

Cited By ~ 13

Author(s):

G. Nicolas ◽

F. Gaill ◽

L. Zylberberg

Keyword(s):

Osmium Tetroxide ◽

Type I Collagen ◽

Freeze Substitution ◽

Immunogold Electron Microscopy ◽

Type I ◽

Fish Scale ◽

Electron Microscopic ◽

Lr White ◽

Fibrillar Collagens ◽

Freeze Fixation

Two fibrillar collagens, the worm cuticular collagen and the vertebrate Type I fish scale collagen, both organized in a compact tissue, were localized by immunogold electron microscopy in resin sections after freeze-fixation and freeze-substitution. Identification of these two fibrillar collagens failed with the-use of postembedding labeling after conventional electron microscopic processing. Positive labeling of the Type I collagen was observed in sections of fish scales freeze-fixed by either slam-freezing or high-pressure freezing, freeze-substituted in acetone with or without osmium tetroxide, and embedded in LR White. The worm cuticular collagen was detected in sections of cuticle that were freeze-fixed, freeze-substituted (necessarily with osmium tetroxide added to acetone), and embedded in either LR White or Epon. It was also detected in specimens pre-fixed by aldehydes before freeze-fixation. The Type I fish scale collagen appears to be more sensitive than the fibrillar cuticular collagen of worms to the procedures employed for postembedding immunoelectron microcopy. Our results have shown that freeze-fixation and freeze-substitution preserved the antigenicity of the fibrillar collagens organized in a compact three-dimensional network, whereas immunolabeling failed after conventional electron microscopic procedures. These cryostabilization techniques appear to be of value to improve the immunolocalization of collagens.

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