Enzo Life Sciences provides more than 40 years of experience in the manufacturing and supply of research kits, biochemicals and biologicals. As Scientists Enabling Healthcare, we are happy to provide simple but useful tips for improving daily tasks as well as the overall quality of your research. With this in mind, here is a comprehensive list of tips for achieving high quality data by in situ hybridization, a widely used biological technique providing the end user with accurate localization of endogenous, bacterial or viral nucleic acids such as DNA, mRNA, and microRNA in metaphase spreads, cells, and tissue preparations.

1. Suitable Probe

As hybridization can take place between complementary deoxyribonucleotides or ribonucleotides, either DNA- or RNA-based probes (riboprobes) can be used to localize DNA or RNA in a given sample. However, RNA-RNA hybrids are more stable than RNA-DNA hybrids, which in turn are more stable than DNA-DNA hybrids. Double-stranded DNA probes are easy to prepare, label, and work with in the laboratory, while single-stranded RNA probes are uniform in size, achieve high incorporation of label, and form highly stable RNA-RNA hybrids. Also, single-stranded oligonucleotide probes can be chemically synthesized and labeled to high specific activity, though RNA probes need to be handled carefully due to the labile nature of RNA. For more information on choosing between DNA- or RNA-based probes, please refer to the TechNote “DNA vs. RNA Probes”. Alternative peptide backbones or locked nucleic acids (LNA) are available today as well, which may enhance hybridization efficiency and stability.

2. Appropriate Label

If the aim is to visualize the probes in combination with the surrounding cells or tissue, which is most often the case, you may choose between a wide range of fluorescent dyes that allow for direct detection of the probes. Alternatively, you could also use intermediaries, such as biotin or digoxigenin. These tags will then be exploited by the detection system (e.g. antibodies conjugated with a reporter enzyme). It is important to have a good level of incorporation of the labelled nucleotide and a proper labelled/unlabelled nucleotide ratio in order to have specific and clear staining that can endure the stress of time, much like immunohistochemistry (IHC).

3. Correct Labeling Technique

Nick translation or random-primed labeling are the methods of choice for generating long, double-stranded DNA probes, while in vitro transcription from vectors containing RNA polymerase promoters is used for the production of riboprobes.

4. Appropriate Detection Method

Direct detection is possible due to fluorescent labels that can be introduced during FISH probe synthesis and detected by fluorescence microscopy. This way, multiplexing can easily be envisaged, as two or more different probes labeled with different fluorophores can be visualized at any single time. Biotin labels can be detected with avidin from egg white or streptavidin from the bacteria S. avidinii (e.g. SAVIEW® PLUS reagents) while digoxigenin can be paired with anti-digoxigenin antibodies (e.g. DIGX® linkers). Both indirect labeling methods can finally be visualized with either alkaline phosphatase (AP) or horseradish peroxidase (HRP), which can react with specific substrates (e.g. HIGHDEF® IHC chromogens) to produce a chromogenic precipitate for CISH.

5. Optimize Proteinase K Digestion

Proteinase K digestion is a critical step for successful ISH as insufficient digestion will result in a diminished hybridization signal. On the other hand, if the sample is over digested, tissue morphology will be poor or completely destroyed, making localization of the hybridization signal impossible. Therefore, optimal assay concentrations of Proteinase K will vary depending upon the tissue type, length of fixation, and size of the tissue. In general, a good starting point for ISH applications is the use of 1-5 µg/mL Proteinase K for 10 minutes at room temperature. Determination of the optimal Proteinase K digestion conditions should be done by a Proteinase K titration experiment followed by hybridization with the probe of choice. The Proteinase K concentration that produces the highest hybridization signal with the least disruption of tissue or cellular morphology is the one you should choose for your assays.

6. Enhance Hybridization Conditions

The aim of ISH is to obtain hybridization only between the probe and its target and thus to achieve the highest specificity possible. Hybridization specificity (stringency) is primarily driven by the degree of homology between the probe and target nucleic acid sequences, probe concentration, temperature and time of hybridization, and concentration of monovalent cations present in the hybridization solution. In particular, hybridization temperature, typically ranging between 37°C and 65 °C, should be optimized carefully for achieving highly specific results. Formamide allows hybridization at temperatures significantly lower than the actual melting temperature of a probe-target-hybrid and thus may assist in the conservation of the morphology of samples.

7. Optimize Post-hybridization Conditions

The reactions are followed by post-hybridization washes of increasing stringency to dissociate imperfect matches, which leaves only specifically bound probe on target sequences. If you are struggling with high background in your ISH assays, you can further reduce background by digesting non-specifically bound probes with nucleases. Use SA nuclease (single-strand-specific endonuclease) for DNA probes and RNase A (endo-ribonuclease degrading ssRNA) for RNA probes before proceeding to the detection step.

8. Modify Washing Steps if Using DNA Probes

DNA probes can provide equivalent sensitivity to RNA probes. However, DNA probes do not bind as tightly to the target molecules in your samples, therefore, formaldehyde should not be used in the post hybridization washes when using DNA probes. Washing steps can be optimized by adjusting temperature, salt, and detergent concentration in the wash buffer to minimize non-specific interactions.

9. Optimize Detection

When using biotinylated probes, be aware that biotin is also endogenously produced in cells and tissues. Endogenous biotin may lead to non-specific staining as avidin- and streptavidin-based detection systems will bind to all biotin molecules that are freely accessible in your samples. In those cases, you either need to block endogenous biotin by adding excess avidin or streptavidin to your samples prior to probe hybridization or use digoxigenin instead of biotin as your probe label. Digoxigenin is a non-radioactive immune tag isolated from the Digitalis plant and as such, is unlikely to be detected by biological materials other than specific anti-digoxigenin antibodies. Hence, use of a digoxigenin label allows for probe detection with high affinity, sensitivity, and most importantly, specificity.

10. Change Reagents Frequently

In order to get the most reproducible and valid data, triethanolamine and acetic anhydride should be replenished once every two to three weeks and 10% neutral buffered formalin (NBF) should be changed out every three to four days.
Enzo Life Sciences is a recognized global leader in providing DNA and RNA labeling technologies with several key patents in developing biotin, digoxigenin, and fluorescent labeled nucleotide probes for gene- and gene expression-related studies. We offer a broad range of products for in situ hybridization (Fig. 1).

Figure 1: Enzo offers a complete set of solutions for in situ hybridization, providing everything you need for labeling, hybridization, and detection.

For a simple and efficient method for generating labeled DNA, please check out our Nick translation DNA labeling kit, Bio-16-dUTP, digoxigenin-dUTP, as well as our SEEBRIGHT® fluorescent dye-dUTPs. For visualizing the expression and spatial localization of target genes, please have a look at our AMPIVIEW™ RNA probes webpage for further information. Enzo can design probes for practically ANY gene in ANY genome to be used in ANY tissue or cells. To order or inquire about custom probes, please fill in the form and a product specialist will contact you. For all questions and concerns regarding any of our products, our Technical Support Team is here to assist.

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