Medicilon’s ISH (In Situ Hybridization) Service

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., 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, in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

ISH is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding the organization, regulation and function of genes. The key techniques currently in use include: ISH to mRNA with oligonucleotide and RNA probes; analysis with light and electron microscopes; whole mount ISH; double detection of RNAs and RNA plus protein; and fluorescent ISH to detect chromosomal sequences. DNA ISH can be used to determine the structure of chromosomes.

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. 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 (i.e. only exact sequence matches will remain bound). Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases is localized and quantified in the tissue using 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.

Different techniques of ISH including Genomic ISH techniques: mainly using the difference of DNA homology between species, with another species of genomic DNA at the appropriate concentration for blocking, in the target chromosome ISH; Fluorescence ISH Techniques; Multi-Color Fluorescence ISH Techniques; Situ PCR etc.