Are you aware of ISH? Well, In Situ Hybridization, commonly known as ISH, is a method that is implemented for detecting certain RNA and DNA sequences in tissue samples by using DNA or RNA probes. Antibodies are used to detect the labeled probes. ISH is considered as one of the most significant techniques that are used vastly in clinical settings these days for scientific research. It is even utilized as the diagnostic tool in comprehending cytogenetics, prenatal diagnosis as well as development, gene expression as well as gene mapping.
The experts detect specific nucleotide sequence by making use of complementary strands that are hybridized with probes. These hybrids are visualized by using autoradiography for the probes which are radioactively tagged. When it comes to the non-isotope labeled probes, the hybrids are determined by the growth of histochemical chromogen. The benefit of making use of ISH enables the researchers to understand the gene sequence of target protein or else gene. To find more about ISH, go through the following write-up.
The two key methods of ISH that are implemented to visualize the DNA as well as RNA are FISH (Fluorescent ISH) and CISH (Chromogenic ISH). Nucleic acid probes are used in both the methods that are hybridized along with tissue samples. The visualization of products is done by using either fluorescent microscopy or bright field microscopy. The preparation of the tissues, as well as the methods implemented to add certain nucleotide probes, are alike in both CISH and FISH. What’s different in both these procedures is the probe detection method. The probes that are used in these techniques range from centromere repeats to whole chromosomes and lot more.
- The tissues used in the procedure are either paraffin or else frozen embedded tissues. At times, the experts also make use of Formalin-fixed tissues.
- Tissues are generally cut to the size of .4 -.5 µm. These tissues are then loaded on slides.
- These tissues are then absorbed by proteinase K in order to enhance hybridization signal. It eliminates the proteins around targeted nucleotide sequence.
- The sample is washed thoroughly with ethanol and xylene.
Addition of Probe
- The kinds of probes that are used in the procedure are cRNA, cDNA as well as synthetic oligonucleotides.
- The DNA probe hybridizes with the corresponding cDNA and the RNA probe with the corresponding mRNA.
- The selection of probes mostly depends on the specificity as well as sensitivity of the probe.
- These probes are almost 40 to 1000 bases long.
- These probes are categorized either with a non-isotope or else an isotope.
- Denaturation mostly results at 95° C.
ISH allows numerous hybridizations to maximize the usage of tissue owing to the availability of finite amount of samples from embryonic tissues and biopsies. The experts also use frozen tissue samples for further research. Certain developments are continually being made for enhancing the sensitivity of this ISH technique either by amplifying target nucleotide sequence prior to the procedure or else after the end of hybridization technique.