Fluorescence In Situ Hybridization for Chromatin Mapping

Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100/PBS for 20 min. After washing with PBS, slides were incubated with 100 ug/ml RNase A for 1 h and 40 units/ml pepsin for 10 min at 37 °C, and dehydrated for 2 min each in 70%, 80% and 100% ethanol. Slides were denatured in 70% formamide for 5 min at 73 °C. DNA probes were prepared and labeled with rhodamine-dUTP and fluorescein-dUTP. Probes were denatured for 5 min at 73 °C and hybridized to the denatured slides for 24 h at 37 °C. Slides were washed for 5 min each in 50% formamide/2x SSC and 0.1% Tween 20/2x SSC at 42 °C, dehydrated for 2 min each in 70%, 80% and 100% ethanol, and mounted on glass slides. FISH signals were detected by a Zeiss imager Z1 Apotome Microscope. DNA probes used in these assays were the BAC RP11–599H8 (155 kb), RP11–942D19 (182 kb), BAC RP11–956H14 (185 kb) and RP11–96K4 (168 kb). Interprobe distances under each condition were calculated as described previously50 (link). 100 cells (200 loci) were used to measure interprobe distances for each condition, and the statistical significance of differences between the indicated groups was measured by the Mann-Whitney U test.