title: Quantitative Immunofluorescence Analysis of Nucleolus-Associated Chromatin.
abstract: The nuclear distribution of eu- and heterochromatin is nonrandom, heterogeneous, and dynamic, which is mirrored by specific spatiotemporal arrangements of histone posttranslational modifications (PTMs). Here we describe a semiautomated method for the analysis of histone PTM localization patterns within the mammalian nucleus using confocal laser scanning microscope images of fixed, immunofluorescence stained cells as data source. The ImageJ-based process includes the segmentation of the nucleus, furthermore measurements of total fluorescence intensities, the heterogeneity of the staining, and the frequency of the brightest pixels in the region of interest (ROI). In the presented image analysis pipeline, the perinucleolar chromatin is selected as primary ROI, and the nuclear periphery as secondary ROI.
authors: Biochemistry Center Regensburg, University of Regensburg, Universitaetsstr. 31, 93053, Regensburg, Germany. stefan.dillinger@ur.de.
authors: Stefan
authors: S
authors: Dillinger
authors: Biochemistry Center Regensburg, University of Regensburg, Universitaetsstr. 31, 93053, Regensburg, Germany. attila.nemeth@ur.de.
authors: Attila
authors: A
authors: Németh
mesh_terms: D002466:Cell Nucleolus / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D011499:Protein Processing, Post-Translational
chemical_list: D002843:Chromatin; D006657:Histones
keywords: Chromatin; Histone posttranslational modification; ImageJ; Nuclear architecture; Nuclear periphery; Nucleolus; Quantitative immunofluorescence