Fixation of the central nervous system by perfusion with aldehydes and its effect on the extracellular space as seen by electron microscopy

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Abstract
Rat brains fixed by perfusion with glutaraldehyde and prepared for electron microscopy under certain conditions showed an abundance of very closely apposed or completely fused surface membranes in some areas. This apparent lack of extracellular space was most consistently observed in the lateral geniculate body and the superior colliculus and less consistently in the cerebellum and parietal cortex. Lowering the temperature of the perfusate from body temperature (38–39 °C) to room temperature (19–20 °C) and/or lowering the pressure at which it was delivered (from 140 to 70 cm of water) led to the appearance of more extracellular space. A striking change in the amount of extracellular space was observed when ethanol was used for dehydrating the samples instead of acetone. When ethanol was employed a space of approximately too A was seen between most cellular elements, whereas acetone dehydration led to an abundance of closely apposed or fused surface membranes. It is suggested that if glutaraldehyde acts by cross-linking proteins in apposing membranes, this reaction is more effectively completed in the presence of acetone and that this may be due to depolymerization of glutaraldehyde by the solvent. It is also suggested that the variation in the amount of extracellular space seen in brains fixed by perfusion with aldehydes may reflect real differences in distances between membranes at the time of fixation.