GUTH 237convincingly by both light and electron microscopy, the importance
of Windle’s principles of perfusion fixation. Thanks in large measure to
the pioneering research in the Laboratory of Neuroanatomical Sciences,
perfusion fixation became the accepted standard of tissue preservation
for both light and electron microscopy.
Palay
At the time of Palay’s arrival, most electron microscopists fixed their
specimens by immersion in osmium tetroxide solution. Because of the
poor penetration of osmium tetroxide, this procedure fixed only the ex
ternal surface of the specimens and left the bulk of the specimen unusable.
Many years earlier, while a postdoctoral fellow in Ernst Scharrer’s labo
ratory at Case Western Reserve University School of Medicine, Palay had
learned about the importance of perfusion fixation for light microscopy
from Scharrer. Now, with further encouragement from Windle, Palay set
about developing a method of perfusion fixation for the electron
microscopical examination of nervous tissue. His first success came when
he adopted a modification of Windle’s two-step procedure: he perfused
the vascular system with a balanced salt solution to remove all traces of
blood and followed this by perfusion with a solution of osmium tetrox
ide to fix the tissue. Although this procedure was a vast improvement over
immersion fixation with osmium tetroxide, the fixative was very costly
and, being highly volatile and caustic, required special precautions to avoid
damaging the investigator’s cornea and respiratory passages. The success
of Palay’s studies led numerous scientists world-wide to attempt further
modifications that might obviate these problems. A procedure involving
three successive steps was developed that soon became standard: (1) removal
of blood by perfusion with an isotonic salt solution; (2) fixation of the
tissues by perfusion with an aldehyde fixative (such as acrolein or a reagent
grade formaldehyde that was freshly prepared from paraformaldehyde);
and (3) post-fixation by immersion of the specimen in osmium tetroxide.
These improved methods of tissue fixation enabled Palay to perform
his pioneering ultrastructural investigations of neurons and neuroglia. His
papers on the ultrastructure of the synapse^10 delineated for the first time
the synaptic cleft, synaptic vesicles, and the various presynaptic and post
synaptic membrane specializations. This description of the ultrastructure