Further observations on the morphological alterations of the glomerular capillary wall of the rat kidney caused by chemical and physical agents: standard procedures versus quick-freezing and freeze-substitution.

The purpose of the present study was to pin-point which of the various preparatory steps required by different histological procedures (conventional, cryosubstitution, freezing followed by cryosubstitution or replication) induces alterations in the fine structure of the endothelial and epithelial surface coat (SC) and of the interposed glomerular basement membrane (GBM). Samples of rat kidney cortex prefixed by vascular perfusion, and isolated glomeruli fixed by immersion, were used. The results demonstrate a continuous, homogeneous and amorphous SC and a GBM devoid of laminae rarae when freezing is used and followed either by cryosubstitution and embedding or by fracture, deep etching and replication. Postfixation in OsO4, and especially dehydration in organic solvents at room temperature, generate a filamentous SC and a GBM with laminae rarae. The different morphology may be due to an extraction of glycoconjugates from SC and GBM during postosmication and/or especially to precipitation of these components during dehydration by organic solvents at room temperature. Studies on isolated glomeruli show comparable results and, in addition, demonstrate that the distance of the capillary loop from the surface of the block influences the morphology of SC and GBM. Rinsing the vascular bed before isolation does not induce a loss of SC or GBM components.