The ISH in FISH – What is In Situ Hybridization?
In situ hybridization is a powerful scientific research tool used primarily for analyzing DNA organization (DNA ISH or DNA FISH) and RNA expression (RNA ISH or RNA FISH) in tissue samples.
As defined by NCBI, “In Situ Hybridization (ISH) is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic section. The underlying basis of ISH is that nucleic acids, if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid to which a reporter molecule is attached.”1 DNA ISH or RNA ISH can be used to study DNA or RNA molecules using this technique respectively.
Two of the more common uses of ISH actually incorporate fluorescence for detection and analysis:
RNA FISH: “Fluorescent in situ hybridization targeting ribonucleic acid molecules (RNA FISH) is a methodology for detecting and localizing particular RNA molecules in fixed cells. This detection utilizes nucleic acid probes that are complementary to target RNA sequences within the cell. These probes then hybridize to their targets via standard Watson–Crick base pairing, after which one may detect them via fluorescence microscopy, either through direct conjugation of fluorescent molecules to the probe or through fluorescent signal amplification schemes.”2
DNA FISH: “FISH tethers fluorescent molecules to defined sections of DNA, making those sections glow under the right wavelength of light. It is possible to collect images of the fluorescent DNA regions under a microscope to see where they are in relation to each other and to the rest of the cell.”3
Formaldehyde fixation (typically FFPE or Formalin-Fixed Paraffin-Embedded) has often been used to preserve and stabilize tissue samples for DNA FISH, RNA FISH, DNA ISH, and RNA ISH analysis. CellCover is a non-toxic alternative to formaldehyde fixation which liquid freezes tissue samples for these analyses. Cells maintain close to in vivo morphology without chemical crosslinking of biomolecules, which also allows for straight forward DNA or RNA isolation from the original tissue sample if subsequent analyses such as next generation RNA sequencing or microarray analysis are required.
To learn more about CellCover as a formaldehyde fixation alternative for in situ hybridization experiments,
2Raj A. (2013) Single-Molecule RNA FISH. In: Roberts G.C.K. (eds) Encyclopedia of Biophysics. Springer, Berlin, Heidelberg
3Fields B. D., Nguyen S. C., Nir G., Kennedy S. (2019). A multiplexed DNA FISH strategy for assessing genome architecture in Caenorhabditis elegans. ELife 8. 10.7554/eLife.42823.030. https://elifesciences.org/articles/42823