IMMUNOLOCALIZATION OF TYPE-III COLLAGEN IN HUMAN ARTICULAR-CARTILAGE PREPARED BY HIGH-PRESSURE CRYOFIXATION, FREEZE-SUBSTITUTION, AND LOW-TEMPERATURE EMBEDDING

We localized Type III collagen by immunogold electron microscopy in re sin sections of intact normal and osteoarthritic human articular carti lage. Comparisons of antibody staining between tissue prepared by high -pressure cryofixation and freeze-substitution without fixatives and t hat exposed to conventional mild chemical fixation with paraformaldehy de 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 det ected with two polyclonal antibodies, one against the triple-helical d omain of the molecule and a second against the more antigenic, globula r amino pro-peptide domain, which in this collagen is retained in the extracellular matrix after secretion. Positive labeling was seen in as sociation with the major interstitial fibrils, suggesting co-polymeriz ation of Types III and II collagen in cartilage. Type III collagen cou ld not be detected in aldehyde-fixed normal cartilage. In fixed osteoa rthritic cartilage, Type III was detectable only when the antibody to the amino pro-peptide was employed. In contrast, high-pressure cryofix ation and freeze-substitution preserved epitopes for both antibodies, permitting immunodetection of Type III collagen in normal and osteoart hritic cartilage. Cryotechniques offer exciting possibilities for sign ificantly improving the immunolocalization of collagens and other fixa tive-sensitive antigens in situ.