3D-FISH on Cultured Cells Combined with Immunostaining
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.
Key wordsInterphase nucleus Cultured cells 3D-preservation of nuclear morphology Fluorescence in situ hybridization (FISH) 3D-FISH Immunostaining Immuno-FISH
The authors thank Jeff Craig and Andy Choo (Murdoch Childrens Research Institute, Australia) for providing BAC RP11-113G13 DNA and anti-CENPA serum. This work was supported by DFG grants SO1054/1 (to IS) and CR59/28 (to MC).
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