In situ hybridization (ISH) is a powerful technique used in molecular biology to visualize the spatial distribution of specific RNA molecules within cells or tissues. To carry out a successful ISH experiment and troubleshoot any potential issues, it is essential to understand the individual steps involved in the protocol. In their fantastic review with the title “A technical review and guide to RNA fluorescence in situ hybridization” Young et al. 2020 PeerJ discuss critical aspects of hybridization experiment.
The authors discuss the technique of RNA-fluorescence in situ hybridization (FISH), which is used to visualize messenger RNA transcripts in cells, tissues, or whole-mount preparations. Over time, various protocols for this technique have been published, offering multiple options for tissue preparation, hybridization, and background removal. The review covers common methods for different sample types, reagents used in tissue preparation and washing, probe types, necessary controls for accurate gene expression visualization, and recent advances in FISH technology.
The authors briefly discuss the evolution of Fluorescence In Situ Hybridization (FISH) techniques, ranging from radioactive to fluorophore-based probes, including single-molecule FISH (smFISH), highlighting the complexity of FISH protocols in the process, before diving into the details.
Probe design
Fluorescence In Situ Hybridization (FISH) probes are nucleic acid strands made of DNA, cDNA, or RNA, ranging from 20 to 1,500 bases in length. To ensure proper hybridization, the probe sequence must complement the target sequence. Probes can be modified with a directly attached fluorophore for detection via fluorescence microscopy or linked to an antibody bound to an antigen in the probe. There are two common types of probes: riboprobes, which are single-stranded RNA probes of 500-1,500 bases generated through in vitro transcription of a cloned target sequence, and oligonucleotide probes, consisting of short single-stranded synthetic DNA labeled with fluorophores. Riboprobes are cost-effective and easily produced, allowing post-hybridization RNase treatment to reduce background. Oligonucleotide probes offer high specificity, efficient tissue penetration, and facilitate additional probe hybridization. However, their production can be challenging and expensive, whether done in-house or outsourced to commercial suppliers.
Tissue preparation and permeabilization
The authors describe the intricacies of tissue preparation, a fundamental aspect of Fluorescence In Situ Hybridization (FISH) protocols. Tissue preparation in FISH involves fixation and permeabilization, critical steps that significantly influence the success of the experiment. Fixation is essential to preserve the tissue’s morphological integrity and minimize enzymatic degradation over time. Common fixatives include formaldehyde or paraformaldehyde (PFA) in phosphate-buffered saline (PBS). These fixatives form covalent links between macromolecules, creating a mesh within cells or tissues that holds their components in place.
Fixation protocols vary based on the sample type. For instance, planarian worms achieve optimal fixation with 4% formaldehyde for 20 minutes, while bacterial species or eukaryotic cells might require fixation for as little as 10 minutes or as long as 90 minutes. The fixation duration depends on factors such as sample size and density, with larger and denser samples requiring longer fixation times. Zebrafish embryos and the annelid Platynereis dumerilii can be suitably fixed in 4% PFA for 2 hours at room temperature or overnight at 4°C. Ethanol and methanol, alcohol-based fixatives, replace free water in tissues, dehydrate cells, and destabilize hydrophobic and hydrogen bonds. These fixatives are commonly used for cell cultures and are effective in as little as 10 minutes.
Permeabilization following fixation allows hybridization reagents to penetrate the tissue effectively. Detergent treatment, commonly using Tween-20, disrupts cellular membranes, enhancing tissue permeability. Whole-mount preparations often require stronger detergent treatments, such as 4% Triton X-100, to ensure complete permeabilization. Additionally, proteinase K treatment can permeabilize tissues and release nucleic acid molecules from bound proteins, making them accessible for hybridization. The degree of permeabilization is crucial, requiring careful optimization, as inadequate digestion can prevent probe penetration, while excessive digestion can disrupt tissue morphology and increase background noise.
Alternative or additional permeabilization methods include treatments with HCl, Triton X-100 addition during fixation, hydrogen peroxide (H2O2) treatment, or the use of organic solvents like acetone. The choice of permeabilization method depends on the sample type and the specific requirements of the experiment. Balancing the fixation strength, duration, and temperature with proteinase-based permeabilization methods is essential to achieve a consistently high signal-to-noise ratio in FISH experiments. Overall, meticulous optimization of these steps ensures the success and accuracy of FISH procedures.
Note: The authors list the fixation time for a whole mouse brain as “6 h at room temperature or over night at 4˚C”. However for RNA detection this is fixation method is too slow. I would recommend a perfusion fixation for mouse brains.
Hybridization
Crucial components and conditions are required for efficient and complete hybridization in Fluorescence In Situ Hybridization (FISH) experiments. The hybridization reaction involves various components such as saline-sodium citrate buffer (SSC), formamide, dextran sulfate, bovine serum albumin (BSA), competitor tRNA or DNA, and the probe. Formamide, a key component, reduces the free energy of binding of nucleic acid strands, allowing hybridization at lower temperatures without losing specificity.
Dextran sulfate, an anhydroglucose polymer, reduces free water in the reaction, forcing the probe and target closer together, enhancing the rate of hybridization (molecular crowding effect). BSA is used as a blocking agent to reduce background signal by blocking nonspecific binding of probe molecules to nucleic acid binding sites on proteins within the tissue. Competitive nucleic acids like tRNA saturate nonspecific binding sites, reducing background and protecting target mRNA molecules.
Hybridization conditions, including salt concentration, pH, hybridization temperature, and duration, must be optimized. The stringency of hybridization, essential to prevent nonspecific hybrids, is affected by salt concentration and hybridization temperature. Generally, hybridization temperatures for short oligonucleotide probes (20-50 nucleotides) are around 37°C, while longer riboprobes (1,000+ nucleotides) may require temperatures >55°C. Hybridization duration of 12-24 hours is typical.
Attention to these parameters and meticulous optimization are crucial to establish the ideal hybridization environment, ensuring specific and accurate results in FISH experiments.
Note: The authors list Vanadyl-Ribonucleoside Complex (VRC) an RNase inhibitor as a component of the hybridization solution. This is not strictly necessary given the presence of Formamide.
Stringency Washes
The post-hybridization washes in Fluorescence In Situ Hybridization (FISH) experiments aim to remove nonspecific hybrids and unbound probe molecules, minimizing background signal. Stringency in these washes is increased through sequential washes with reduced salt concentrations, matched to the hybridization temperature, preserving specific labeling while minimizing nonspecific hybrids. Issues like autofluorescence and excessive background can be tackled using methods like treatment with 0.1% Sudan Black B in 70% ethanol to reduce autofluorescence or acetylation with 0.3% acetic anhydride in triethanolamine to block positively charged proteins. Before visualization, tissue clearing methods prevent light scattering within the tissue, including organic solvent-based methods, formamide-based ClearT, and urea-based CUBIC. Advanced techniques like using anchor probes in a polymer matrix are suitable for highly multiplexed FISH experiments but require careful consideration.
Note: I would like to include for the buffer preparation nuclease-free water should be used for best results.
Controls for FISH experiments
Careful design of controls is crucial in FISH experiments to differentiate genuine biological signals from false positives or technical issues. Positive controls, like widely expressed genes or tissue-specific markers, help verify protocol efficacy. Negative controls, such as RNase-treated samples or sense probes, identify nonspecific binding. Combining these controls and considering strand-specific transcription patterns enhances specificity and aids in distinguishing background noise from actual biological signals.
In conclusion, FISH is a powerful technique to study gene expression patterns in biological systems at various scales. Combined with other molecular methods, it enhances genetic understanding in unconventional model organisms.
This review outlines key FISH principles, emphasizing crucial steps like fixation with 4% PFA, permeabilization, and hybridization. Post-hybridization washes with formamide and Tween-20 at decreasing salt concentrations remove nonspecific hybrids. Head over to the full text of the article for more details.
Young AP, Jackson DJ, Wyeth RC. A technical review and guide to RNA fluorescence in situ hybridization. PeerJ. 2020;8:e8806. Published 2020 Mar 19. doi:10.7717/peerj.8806
Biology icons created by Freepik – Flaticon
Featured image generated via imagine.art
insitutech 
Leave a comment