Chromosome structure: improved immunolabeling for electron microscopy

To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIalpha (topoIIalpha) and condensin. Immuno-electron microscopy (EM) of thin-sectioned chromosomes is the method of choice toward this goal. A much-improved immunoprocedure that avoids problems associated with aldehyde fixation, such as chemical translinking and networking of chromatin fibers, is reported here. We show that ultraviolet irradiation of isolated nuclei or chromosomes facilitates high-level specific immunostaining, as established by fluorescence microscopy with a variety of antibodies and especially by immuno-EM. Ultrastructural localizations of topoIIalpha and condensin I component hBarren (hBar; hCAP-H) in mitotic chromosomes were studied by immuno-EM. We show that the micrographs of thin-sectioned chromosomes map topoIIalpha and hBar to the center of the chromosomal body where the chromatin fibers generally converge. This localization is defined by many clustered gold particles with only rare individual particles in the peripheral halo. The data obtained are consistent with the view that condensin and perhaps topoIIalpha tether chromatin to loops according to a scaffolding-type model.