Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.