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A comparison of freeze-substitution with other methods for preservation of the pulmonary alveolar lining layer.

Rapid freezing of expanded lungs and freeze-substitution in acetone containing OsO4 partially preserved the pulmonary alveolar lining layer for electron microscopy. The lining layer consisted of granular or flocculent material with a surface film at its interface with the air. It filled the spaces between protruding capillaries and other depressions in the alveolar wall. It is unlikely that this lining material is an artifact resulting from transfer of fluid during freezing because similar results were obtained after fixation by vascular perfusion and by immersion of the intact, air-filled lung in aqueous fixatives containing glutaraldehyde.