Chromosome structure: improved immunolabeling for electron microscopy

Article Article scientifique Article Chromosome structure: improved immunolabeling for electron microscopy eng 2005 author Maeshima Kazuhiro 130365 author Eltsov Mikhail 531650 author Laemmli Ulrich Karl 25441 365-375 10.1007/s00412-005-0023-7 0009-5915 To structurally dissect mitotic chromosomes, we aim to position along the folded chromatin fiber proteins involved in long-range order, such as topoisomerase IIα (topoIIα) 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 topoIIα 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 topoIIα 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 topoIIα tether chromatin to loops according to a scaffolding-type model. Dewey 540 Dewey 570 Open Access - Licence nationale Springer 39 Département de biochimie 144 43 Département de biologie moléculaire 161 false Chromosoma 114 5 5db6f0a6-9cd4-4ec7-b97e-77fbd20e25b2 Article (Published version) Public Maeshima2005_Article_ChromosomeStructureImprovedImm.pdf 514218 application/pdf openAccess Faculté des sciences / Section de chimie et biochimie / Département de biochimie Faculté des sciences / Section de biologie / Département de biologie moléculaire

Title	Published in	Access level	OA Policy	Year	Views	Downloads

Chromosome structure: improved immunolabeling for electron microscopy	Chromosoma			2005	399	463

Archive ouverte UNIGE

Eltsov, Mikhail