FLUORESCENCE IN-SITU HYBRIDIZATION OF OLD G-BANDED AND MOUNTED CHROMOSOME PREPARATIONS

An improved method for fluorescence in situ hybridization (FISH) inves tigation of old, previously G-banded, mounted chromosome preparations with chromosome specific painting probes and centromere-specific probe s is described. Before hybridization, the slides are incubated in xyle ne until the coverslips detach spontaneously; any mechanical manipulat ion will jeopardize the results. The success of chromosome painting is improved by excluding the regular RNase treatment step prior to hybri dization. Additional changes compared with standard FISH protocols are that the 2 x SSC step is omitted, that the amount of added probe is i ncreased approximately 2.5 times, and that the amplification of signal s is performed twice. The applicability of the method, which allows do uble painting with two differently labeled probes using two differentl y fluorescing colors, was tested on 11 cases involving different chrom osome abnormalities and different types of material, including short-t erm cultures of epithelial and mesenchymal tumors, blood, leukemic bon e marrow, and long-term cultures of a cell line derived from an epithe lial tumor. Success was achieved even with chromosome preparations tha t were several years old. (C) Elsevier Science Inc., 1997.