Everything about In Situ Hybridization totally explained
In situ hybridization (ISH) is a type of
hybridization that uses a labeled
complementary DNA or
RNA strand (for example,
probe) to localize a specific DNA or RNA sequence in a portion or section of
tissue (
in situ), or, if the tissue is small enough (for example plant seeds,
Drosophila embryos), in the entire tissue (whole mount ISH). This is distinct from
immunohistochemistry, which localizes proteins in tissue sections. DNA ISH can be used to determine the
structure of chromosomes.
Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts.
Process
For hybridization histochemistry, sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. As noted above, the probe is either a labeled
complementary DNA or, now most commonly, a complementary
RNA (riboprobe). The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of unhybridized, excess RNA probe). Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-identical interactions (for example only exact sequence matches will remain bound). Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases (for example,
digoxigenin) is localized and quantitated in the tissue using either
autoradiography,
fluorescence microscopy or
immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.
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