Alterations in the morphology of glycoconjugate molecules caused by histochemical procedures: comparison of renal glomeruli and articular cartilage.

The fine structure of the glycoconjugate molecules was investigated in the glomerular capillary wall of the rat kidney fixed by vascular perfusion, and in the human and rat articular cartilage fixed by immersion. Kidney and cartilage were either prefixed in aldehyde alone (group a), or with the addition of Alcian Blue 8 GX (group b), or Alcian Blue and 0.3 M MgCl2 (group c), or Acridine Orange at a low (0.01%) and high (0.1%) concentration (group d). The specimens were postfixed either in OsO4 phosphate or cacodylate, with the exception of some of the samples in group a, for which a solution of potassium ferrocyanide-reduced OsO4 was used (group e). All samples were conventionally dehydrated and embedded in Epon. In addition, some of the tissue samples in group c were cryoprotected, frozen in liquid Freon (-150 degrees C) or in nitrogen slush (-210 degrees C), both postfixed and dehydrated by cryosubstitution, and embedded in Epon (group f). The present investigations demonstrate that some well known extracellular structures such as the laminae rarae of the glomerular basement membrane or the interfibrillar matrix of the articular cartilage can be considerably altered in their morphology by the histological procedures applied. Whereas the precipitated glycoconjugates, as seen after staining with cationic dyes or reduced OsO4 and conventional dehydration, can easily be recognized, the superposition of the extended molecules, as preserved by freezing and substitution, prevents their demonstration in native conformation.