Visualization of proteoglycans and link protein in embryonic chick limb cartilage via cryofixation, freeze-substitution and immunochemical techniques

Chick embryo limb bud cartilage contains a family of proteoglycans, a few of which have been identified ultrastructurally by antibody labelling. Limb bud cartilage from stage 30–34 chick embryos was high-pressure frozen, freeze-substituted and embedded in Lowicryl resin. Sections were treated with polyclonal antibodies for core protein and monoclonal antibodies for chondroitin-6-sulphate and link protein. Label for core protein was demonstrated on both structural matrix and free within the compartmental space. Quantitative analysis indicates that core protein is preferentially localized on electron-dense structural matrix, and that this distribution is uniform between stages 30 and 34. The association of protein epitopes on electron-dense lattice is strongly influenced, rather than a chance observation. Significant quantities of core protein are also located in the free compartments of the cartilaginous lattice. Chondroitin-6-sulphate and link protein were localized predominantly within the compartments of the embryonic lattice. Our data provide convincing evidence that the proteoglycans were immobilized within a microcrystalline matrix of the embryonic compartments. A role for core protein as a stabilizer within the lattice and in the free space where it serves to aggregate polymeric proteoglycans is suggested from our results.