Simultaneous fluorescence immunostaining and FISH—a combined platform for the in situ co-detection of DNA and proteins—is a powerful method for studying chromosomal and nuclear structure, function, behavior, and variation. Following the success of large-scale omics projects, this approach could become a preferred method for validating molecular findings, as biological processes arise from complex, time- and space-dependent interactions among system components. Importantly, the chromosomal set—the karyotype—plays a central role in organizing gene networks and maintaining system-level information. This chapter outlines classic protocols for preparing mitotic and meiotic chromosomal/nuclear slides, as well as performing DNA–protein co-detection. These flexible methods will be essential for advancing research in the post-sequencing era.