In Vivo and In Situ Replication Labeling Methods for Super-resolution Structured Illumination Microscopy of Chromosome Territories and Chromatin Domains.
Recent advances in super-resolution microscopy enable the study of subchromosomal chromatin organization in single cells with unprecedented detail. Here we describe refined methods for pulse-chase replication labeling of individual chromosome territories (CTs) and replication domain units in mammalian cell nuclei, with specific focus on their application to three-dimensional structured illumination microscopy (3D-SIM). We provide detailed protocols for highly efficient electroporation-based delivery or scratch loading of cell impermeable fluorescent nucleotides for live cell studies. Furthermore we describe the application of (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU) for the in situ detection of segregated chromosome territories with minimized cytotoxic side effects.
Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Ezequiel
E
Miron
Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Cassandravictoria
C
Innocent
Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Sophia
S
Heyde
Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. lothar.schermelleh@bioch.ox.ac.uk.
Lothar
L
Schermelleh
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure*; D020090:Chromosome Segregation; D033481:Chromosomes, Mammalian / Q000235:genetics / Q000648:ultrastructure*; D004261:DNA Replication; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D008856:Microscopy, Fluorescence; D018741:Replication Origin
D002843:Chromatin
Chromatin; Chromosome territories; F-ara-EdU; Replication domains; Replication labeling; Structured illumination microscopy; Super-resolution imaging