Simultaneous production of R-bands and either replication patterns or sister chromatid differentiation

The dye triplet chromomycin/methyl green/DAPI and some related dye combinations were applied to bromodeoxyuridine (BrdU)-substituted and non-substituted chromosomes. In both kinds of chromosome preparations, tri-staining followed by observation at pH 7 resulted in well defined R-bands (excitation wavelength 436 nm) and a mixed DA-DAPI/Q-banding pattern (360 nm). Two approaches have given satisfactory expression both of reverse bands and of the differential BrdU-substitution present in metaphase chromosomes: (1) direct tri-staining at pH 7 and mounting of the preparations at pH 11; or (2) pretreatment of the preparations with Hoechst 33258 plus UV and hot buffer followed by tri-staining and mounting at pH 7. These methods should prove useful for routine chromosome analysis and, in combination with BrdU-labelling, in studies of chromosome structure and replication.