Ultrastructural analysis of nuclear bodies using electron microscopy

Abstract

Recent immunofl uorescent (IF) studies have discovered a variety of nuclear foci that have no known ultrastructurally defi ned counterpart. Using antibodies as ligands, immuno-electron microscopy (I-EM) is the method of choice for high-resolution recognition of these newly described nuclear compartments. However, noncoding RNAs (ncRNAs) have also been shown to be frequent components, sometimes essential, of nuclear bodies so that electron microscopic in situ hybridization (EM-ISH) can be used as an alternative means to characterize nuclear foci at the EM level. Among the array of protocols available, Lowicryl embedding of chemically fi xed cells allows for high preservation of both nuclear structures and antigenicity and provides stable cell and tissue samples that can be re-probed whenever new antibodies or probes become available. Rapid and robust protocols are available for both I-EM and EM-ISH postembedding techniques so that they can be combined on the same sections, providing ultrastructural and molecular insights into newly “emerging” nuclear bodies.