Fluorescence in situ hybridization on three-dimensionally preserved nuclei (3D-FISH), in combination with immunocytochemistry and 3D fluorescence microscopy, is a key tool to analyze the functional organization of the interphase nucleus. In the last decade, 3D-FISH on cultured cells has become a routine technique and is now widely used in nuclear biology. This method allows visualization of chromosome territories, chromosome subregions, single genes, and RNA transcripts preserving their spatial positions in the cell nucleus. In many cases, it is desirable to combine 3D-FISH and immunostaining to map DNA/RNA and protein targets in the same cells. Some steps of the FISH procedure, however, may interfere with immunostaining and special efforts have to be done to combine FISH and antibody staining successfully. The protocol suggested in this chapter describes three variants of combined 3D-FISH and immunostaining which have been successfully used in our laboratory for many years.