Chromatide alexa fluor 594 5 dutp

BAC probes (RP23-41J14 (Igh 3ʹ); RP24-316H6 (Igh 5ʹ) (gifts from the Welcome Sanger Institute, Hinxton, UK); RP23-374P12 (Igl 3ʹ); RP23-382P9 (Igl 5ʹ) (Source Bioscience) were labelled using a nick translation kit (Abbott Molecular) according to the manufacturer instructions, incorporating either Chromatide Alexa Fluor 594-5-dUTP (Thermo Fisher Scientific), Chromatide Alexa Fluor 488-5-dUTP (Thermo Fisher Scientific), Gold-dUTP (Abbott Molecular) or biotin-16-dUTP (Sigma). The probes were resuspended in the presence of a ten times excess of unlabelled mouse C0t1 DNA (Thermo Fisher Scientific), in hybridization buffer (50% formamide, 10% dextran sulphate, 2xSSC), before being denatured for 8 minutes at 85°C, followed by a pre-annealing step 30 minutes at 37°C. The metaphase spreads were denatured in 0.07 N NaOH for 1 minute. The probes were applied onto the slides, and the hybridization was carried out overnight at 37°C. Three post-hybridization washes were performed, in 0.1x SSC buffer at 65°C. Biotinylated probes were detected using streptavidin-Cy5 (Thermo Fisher Scientific). Slides were mounted in Vectashield/DAPI and analysed blind with the microscope described above. Between 98 and 150 metaphases were analysed for each mouse with the exception of one case (45 metaphases collected for 1 control mouse).

Using our published techniques (15 (link), 16 (link), 30 (link)), pre-B cells were processed for 3D DNA FISH. Briefly, probes for 3D FISH were prepared from bacterial artificial chromosomes (BACs). We used RP23-101G13, RP23-26A6, and RP24-387E13, which correspond to the 5′, middle, and the 3′ region of the Igκ locus, respectively. To make probes for each slide, 1 μg BAC DNA samples were labeled by nick translation with ChromaTide Alexa Fluor 488–5-deoxyuridine triphosphate (dUTP), ChromaTide Alexa Fluor 594–5-dUTP (Invitrogen), or Cy5-dUTP (GE Healthcare). Hybridization conditions were as described previously (15 (link), 16 (link), 30 (link)). FISH signals were analyzed by Leica TCS SP5 confocal microscopy with Z slice-sections separated by 0.3 μm, and the center-to-center distances between different hybridizing signals were measured using a plug-in of ImageJ software.