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A Brief Note on the Various Steps of In Situ Hybridization

In situ hybridization (ISH) is used for detecting the RNA and DNA sequences in a sample tissue through the utilization of a labeled DNA or RNA probe. It comprises of two definite techniques- Chromogenic in situ hybridization (CISH) and Fluorescent in situ hybridization (FISH), both of which make use of nucleic acids and the result is visualized via bright field microscopy. The said method is extremely important for it can help one to understand gene mapping, gene expression, cytogenetics, prenatal diagnosis, etc.

In the following write-up, I have chalked down the major steps that are included in ISH. Readers out there are requested to buy some time and take a close look at the below-mentioned pointers.

A Look at the Systematic Process of ISH

Preparing the Tissue

  • The tissues could either be embedded with paraffin or frozen. In certain cases tissues fixed with formalin are also used.
  • After the selection, the tissues are cut and then loaded on the slides.
  • In order to improve hybridization, the tissues are processed with proteinase K. Doing so would remove the unnecessary proteins.
  • The sample is then washed thoroughly with ethanol and xylene.

Probe Addition

  • Three kinds of probes could be used and they are- cRNA, cDNA, and synthetic oligonucleotides.
  • The probes are selected based on their specificity and sensitivity.
  • The probes must be 40 to 1000 bases long and labeled with non-isotope or isotope.
  • Denaturation takes place at a temperature of 95C.


  • In order to increase the rate of hybridization, dextran sulphate is added to the sample.
  • Dithiothreitol (DTT) and formamide are added so that the hybridization can take place at minimum temperatures.
  • Sodium chloride and sodium citrate are mixed and added to the sample so that electrostatic attraction could be alleviated largely.
  • Ethylenediaminetetraacetic acid or EDTA is further added so that divalent cations can be removed from the sample and DNA could be stabilized.
  • The entire process of hybridization takes place at 37C.
  • The samples are then washed at 70C so that loosely bound and excessively bound probes could be removed.

Signal Amplification

  • Probes are joined directly to the various fluorescent molecules such as digoxygenin, biotin, and fluoresceinor indirectly to alkaline phosphate and conjugated antibodies.
  • In case of tyramide signal amplification, the antibody must be bound with horseradish peroxidase. Using HRP would catalyze biotin deposition.
  • DIG and biotin labeled nucleotides are detected indirectly by using antibodies.
  • Fluorescent labels on the other hand are detected by using bright microscope. This device would be used for examining the cells as well as tissues.

Well, the entire process of in situ hybridization can maximize the usage of tissues. Top-notch professionals are continually performing experiments so that the sensitivity of this technique could be enhanced largely.

I am sure this useful content has most certainly allowed all my readers to get a clear idea of in situ hybridization (ISH) and the varied steps included in this particular technique.