Improved preservation of phospholipid‐rich multilamellar bodies in conventionally embedded mammalian lung tissue—an electron spectroscopic study

Abstract

SUMMARY Different conventional methods of tissue processing were studied to determine the extent to which phospholipid‐rich multilamellar bodies of pulmonary alveolar epithelial type II cells of the pig were preserved. Prolonged treatment with half‐saturated aqueous uranyl acetate yielded excellent results on the stabilization of the multilamellar substructure, irrespective of whether glutaraldehyde‐paraformaldehyde or glutaraldehydetannic acid was used as a primary fixative. The lamellar periodicities were observed to be 5·5–6·1 nm. Differences in the phosphorus distribution among several types of lipid bodies of alveolar epithelial type II cells were studied by means of electron spectroscopic imaging and electron energy‐loss spectroscopy. Multilamellar bodies gave phosphorus signals which were significantly higher than those obtained from granular regions of composite bodies, whereas homogeneous bodies gave phosphorus signals which were even lower than those obtained from mitochondria, endoplasmic reticulum membranes or ribosomes.

An Official Research Policy Statement of the American Thoracic Society/European Respiratory Society: Standards for Quantitative Assessment of Lung Structure

“…Embedding in paraffin has some advantages for LM (staining and solubility) and is the traditional standard in pathology (archival material), but causes unpredictable tissue shrinkage, a disadvantage for morphometry unless sampling is done with the fractionator technique aiming at estimation of total number of cells or alveoli (SECTION 4.2.5.). Samples for EM are post-fixed in buffered 1% osmium tetroxide (avoid phosphate buffer here) followed by bloc staining with uranyl acetate solution (61). Dehydration in ethanol series follows before embedding.…”

“…Preservation of surfactant-containing lamellar bodies in alveolar type II cells requires prolonged en bloc staining with half-saturated aqueous uranyl acetate (61,62).…”

Presence of Surfactant Lamellar Bodies in Normal and Diseased Sinus Mucosa

“…The lipid-carbohydrate retention method for preserving lamellar bodies is lengthy and cumbersome [7,8] . We investigated the effi cacy of an alternate preparation technique for electron microscopy to preserve the structure of lamellar bodies and examined a variety of diseased and normal sinus tissues.…”

Electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS) of multilamellar bodies and multilamellar body-like structures in tannic acid-treated alveolar septal cells.

“…After the fixation procedures used in our studies, tannic acid was not observed to improve multilamellar body preservation, which was also reported by Stratton (19). The best results were achieved by en bloc staining in aqueous uranyl acetate overnight (11).…”

“…However, freeze-dried cryosections failed to demonstrate lamellar fine structure (24), although freeze-fracture studies of cryofked Type I1 cells clearly demonstrated the substructure of MLBs seen in conventional preparations (25,26). Since the different types of inclusion bodies in Type I1 alveolar epithelial cells (11,14) …”