In situ hybridization (ISH) is a powerful and versatile technique for localizing and detecting the distribution of specific nucleic acid (DNA or RNA) sequences, relative to their protein products and other cellular components, within a heterogenous cell population. The visualization of precise spatial and temporal genomic loci and gene expression changes within tissues, cells, or chromosomes, makes ISH a very useful tool for biological and diagnostic applications.
In this technique, biological samples make up of tissue sections, cells or chromosomes are affixed to a glass slide and then exposed to a probe — a small piece of single-stranded DNA labeled with a chemical or fluorescent dye. The labeled probe finds and then binds to its matching sequence within the biological sample. The location of the bound probe can then be visualized with a microscope.
Sample Preparation
Tissue fixation and permeabilization are two of the most important aspects of ISH sample preparation, and have a signification impact on the quality of the data extracted. Fixation is typically achieved using 4% paraformaldehyde (PFA), which should be prepared fresh for each usage as PFA can form polymers with long-term storage in solution. An alternative is to use a 1:10 dilution of formalin, which does not need to be prepared fresh each time. The incubation time for fixation is highly variable and dependent on the specific tissues.
Permeabilization is usually achieved after fixation using a detergent such as Tween-20, SDS, or Triton X-100. Detergent concentrations vary from 0.1% to 4% (the bulkier the sample, the more detergent used). For applications other than eukaryotic cells, proteinase treatment is often applied as well, especially when the penetration of oligo probes is considered to be an issue.
Probe Selection
Probes are preselected complementary sequences of nucleotide bases to bind the target DNA or RNA of interest. Probes can be as small as 20-40 base pairs (bp) or up to 1000 bp, although, the optimal size is probably around 50-300 bp.
Many different types of probes can be used include double-stranded DNA probes, single-stranded antisense RNA probes, single-stranded DNA probes generated by polymerase chain reaction (PCR), synthetic oligodeoxynucleotide probes, and oligo-riboprobes. When choosing a probe for ISH, the sensitivity, specificity, tissue penetration, hybrid stability, reproducibility and the application, must be taken into account. Probes are readily available for purchase from many different suppliers who also offer custom-designed probes that are purified and ready to use.
Probe Labeling
There are two main methods for labeling a probe: radioisotope labeling and non-isotope labeling. 3H, 35S, and 32P are widely used radiolabeled probes. The results of radioisotope labeling are easily quantified or semi-quantified by densitometry counting or silver grain counting on film. Problems associated with radioisotope labeling include a long exposure time, poor spatial resolution, risk of exposure to radioactivity, and disposal of radioactive waste. For non-isotope labeling, compounds including biotin, fluorescein, digoxigenin, alkaline phosphatase, or bromodeoxyuridine are used and are visualized by histochemistry or immunohistochemistry. Problems associated with non-isotopic labeling are that it is generally considered not as sensitive as radioactive labeling, and the hybridization results are difficult to quantify.
Denaturation
The next step involves denaturation of the probe and target DNA, which may lead to loss of morphology. A compromise must be found between hybridization signal and morphology. Alkaline denaturation is traditionally used. Heat denaturation is simple and effective, but the time and temperature need to be optimized.
Hybridization
Hybridization is performed by incubating a solution containing hybridization probes with the sample, usually overnight. For ideal hybridization, the reannealing temperature should be set to 20-25°C lower than the melting temperature to generate stable hybridization products. A temperature range of 37-42°C is a good starting point for the hybridization step. The concentration of the probe is also important. Typically, around 300 ng/mL is used, with a range of 200-1500 ng/mL depending on the size of the probe. The hybridization kinetic is also affected by salt concentration. At concentrations below 1.5 M, the higher the salt concentration, the higher the rate of hybridization. Polymers of high molecular weight, such as dextran sulfate and polyethylene glycol, are often added at a concentration of about 10% (v/w). These molecules can create a networking phenomenon that take up sufficient space in the solution to artificially increase the probe concentration and thus the hybridization rate by about tenfold without any noticeable side effects.
Washing
The washing solution should be designed to contain the appropriate salt content to wash away unbound probes and probes in less stable, mismatched hybrids, and leave the perfectly-matched hybrids intact. Washing should be carried out at or close to the stringency condition at which the hybridization takes place with a final low stringency wash. If washing conditions are too stringent, a loss of sensitivity may occur and if they are not stringent enough, there can be high background labeling. The stringency of the washing can be manipulated by varying the formamide concentration, salt concentration and temperature.
Detection & Analysis
Quantitation of the ISH data depends on the type of signal generated by the detection system. For radioactive probes, hybrids are detected auto-radiographically. For non-radioactive probes, the signal is generated directly or indirectly by the labeled probes and detected by microscopy. Since the preparations are permanent, bright-field microscopy is preferred for evaluation of ISH results in most routine applications. However, fluorescence microscopy is highly sensitive and can be used for multiplexing with spectrally different fluorophores.
Advantages and Disadvantages of ISH
A major advantage of ISH is its ability to maximize the use of tissue that is difficult to obtain. In addition, it can be combined with other techniques such as immunohistochemistry to detect protein and mRNA in samples. However, target identification can be difficult using ISH when working with samples with low DNA and RNA copies.