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 (conv entional, cryosubstitution, freezing followed by cryosubstitution or r eplication) induces alterations in the fine structure of the endotheli al and epithelial surface coat (SC) and of the interposed glomerular b asement membrane (GBM). Samples of rat kidney cortex prefixed by vascu lar perfusion, and isolated glomeruli fixed by immersion, were used. T he results demonstrate a continuous, homogeneous and amorphous SC and a GBM devoid of laminae rarae when freezing is used and followed eithe r by cryosubstitution and embedding or by fracture, deep etching and r eplication. Postfixation in OsO4, and especially dehydration in organi c solvents at room temperature, generate a filamentous SC and a GBM wi th laminae rarae. The different morphology may be due to an extraction of glycoconjugates from SC and GBM during postosmication and/or espec ially to precipitation of these components during dehydration by organ ic solvents at room temperature. Studies on isolated glomeruli show co mparable results and, in addition, demonstrate that the distance of th e capillary loop from the surface of the block influences the morpholo gy of SC and GBM. Rinsing the vascular bed before isolation does not i nduce a loss of SC or GBM components.