Fluorescence in situ hybridization techniques for cytogenetic and genomic analyses

Fluorescent in situ hybridization (FISH) is a powerful method to visualize DNA sequences in the context of the whole chromosome. Yet despite the value of FISH analysis for cytogenetic studies, there are surprisingly few labs that are able to adapt the technique for their experiments in chromosomal and genome biology. Here we present a comprehensive FISH protocol acquired from over 20 years of collective experience using different plant species. Our description uses rice as a model for performing a complete FISH procedure, but the protocol can be readily adapted for other plant species. We have provided more specialized instruction beyond routine FISH, which includes the preparation of meiotic and mitotic samples suitable for FISH analysis, procedures for direct and indirect labeling of DNA probes, and techniques for increasing signal strength using layers of antibodies.