Fluorescence in situ hybridization has become a major tool for analysis of gene and chromosome copy number in normal and malignant tissue. The technique has been applied widely to fresh tissue and dispersed formalin-fixed, paraffin-embedded archival tissue, but its use on sections of archival tissue has largely been limited to sections < 6 mu thick. This does not provide intact, uncut nuclei for accurate analysis of gene or chromosome copy number. We report here a method of hybridization to sections > 20 microns thick that overcomes these difficulties. Key developments were the use of DNA probes directly labeled with fluorochromes and optical sectioning using laser-scanning confocal microscopy.