Collagen–proteoglycan interactions. Localization of proteoglycans in tendon by electron microscopy

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Abstract
Proteoglycan in fetal and adult rat tail tendon and adult rabbit achilles tendon was stained for EM with a cationic phthalocyanin-like dye, based on cinchomeronic acid, in a critical electrolyte concentration method. Provided that the tissue was fixed with glutaraldehyde or formaldehyde, regular orthogonal perifibrillar arrays of filamentous material (proteoglycan) were observed, but no intra-fibrillar proteoglycan was seen. Specific proteoglycan-collagen interactions are inferred, and a model is proposed. Without fixation the filamentous arrays disaggregated in the MgCl2 solutions (0.3 M) used during staining. End-to-end proteoglycan aggregation is implied. Tendon and cartilage were compared. Problems of electron-histochemical localization of extended space-filling polyanions by the use of cationic electron-dense precipitants are discussed, particularly polyanion-domain collapse, specificity of staining and fixation. A 2-stage staining procedure that markedly enhances contrast is described. This procedure is based on the multivalent nature of the dye and the consequent anion-exchange properties of the dye-polyanion complex.