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 hybridizat ion of DNA probes and high resolution fluorescent R banding is describ ed. Human lymphocytes were stimulated with phytohemagglutinin and sync hronized using a fluorouracil block followed by exposure to bromodeoxy uridine and Hoechst 33258 prior to harvest. Metaphase preparations wer e treated with Hoechst 33258 and exposed to UV light. Thereafter they were incubated in sodium phosphate buffer and dried prior to in situ h ybridization with a biotin-labelled centromere-specific alpha-satellit e DNA probe for chromosome 1 (pUC1.77) and two digoxigenin-labelled pr obes, i.e., a PCR-generated chromosome 8-specific alphoid probe (# 8) and a cosmid probe for FLT4 gene on 5q33-qter (class III receptor tyro sine kinase). Hybridization signals were detected by an indirect immun ofluorescence method using fluorescein isothiocyanate. The chromosomes were counter-stained with propidium iodide and 4',6-diamidino-2-pheny lindole dihydrochloride. This simple method allows unambiguous chromos ome band identification simultaneously with detection of the hybridize d probes.