Simultaneous Detection of High-Resolution R-Banding and Fluorescence in Situ Hybridization Signals After Fluorouracil-Induced Cellular Synchronization

A method for simultaneous detection of fluorescence in situ hybridization of DNA probes and high resolution fluorescent R banding is described. Human lymphocytes were stimulated with phytohemagglutinin and synchronized using a fluorouracil block followed by exposure to bromodeoxyuridine and Hoechst 33258 prior to harvest. Metaphase preparations were treated with Hoechst 33258 and exposed to UV light. Thereafter they were incubated in sodium phosphate buffer and dried prior to in situ hybridization with a biotin‐labelled centromere‐specific cc‐satellite DNA probe for chromosome I (pUCI.77) and two digoxigeninlabelled probes, i.e., a PCR‐generated chromosome 8‐specific alphoid probe (#8) and a cosmid probe for FLT4 gene on 5q33‐qter (class 111 receptor tyrosine kinase). Hybridization signals were detected by an indirect immunofluorescence method using fluorescein isothiocyanate. The chromosomes were counterstained with propidium iodide and 4.6‐diamidino‐2‐phenylindole dihydrochloride. This simple method allows unambiguous chromosome band identification simultaneously with detection of the hybridized probes.