Optimization of in situ hybridization to human metaphase chromosomes

A simplified method describing optimal conditions for in situ hybridization to human chromosomes is presented. A 1.5-kb DNA fragment coding for part of the 28 S rRNA was subcloned into pSP65. Tritium-labeled RNA was synthesized as runoff transcripts and 39-67% of the labeled probes specifically hybridized to the nucleolar organizer regions of the acrocentric chromosomes. Denaturation performed with 70% formamide, 1 mM EDTA, 2 X SSC gave a high specific hybridization with both fresh chromosome spreads (1-8 weeks) and older preparations (3-6 months). To obtain good chromosome morphology and a high specific hybridization it was important to neutralize the final formamide denaturation mixture containing 70% formamide, 1 mM EDTA, and 2 X SSC, whereas it was unimportant to deionize the formamide. Freshly made slides denatured with 0.15 M NaOH in 70% ethanol hybridized equally well with the rRNA probe. Despite treatment of the chromosomes with RNases before denaturation the following proteinase K and the acetylation steps recommended could be omitted without degradation of the rRNA probe as judged from the high specific hybridization to the nucleolar organizer regions.