Ultrastructural analysis of the extracellular matrix.

Abstract

This chapter gives insight into task-specific methodologies for the evaluation of matrix ultrastructure by light and electron microscopy. It separately considers the isolation and preparation of molecular isolates for negative staining, immunolabeling, rotary shadowing, and single particle analysis. Also considered is the preparation of whole tissues and cultured cells by chemical fixation and cryofixation methodologies. Immunoelectron microscopy for immunoidentification of matrix components may be accomplished en bloc or via section-surface protocols; the advantages and pitfalls in both methodologies are described. Correlative light and electron microscopy, particularly utilizing GFP constructs, demands special consideration in fixation and embedding protocols. TEM imaging methods, including the use of montage software and the acquisition of thick-section tilt series are discussed. The protocols presented in this chapter, with the exception of single particle analysis, are those which are continuously used in our laboratory and represent the latest modifications in our protocols.