ISH: in situ hybridization protocol

Last edited Thu 26 June 2025

Introduction to gene expression analysis

Gene expression analysis is fundamental to understanding how genes function and are regulated within different biological systems. In situ hybridization (ISH) is a powerful technique that enables researchers to visualize the spatial and temporal expression patterns of specific nucleic acid sequences directly within tissue samples or entire tissues (whole mounts). By using ISH, scientists can detect both DNA and RNA sequences, providing insights into where and when particular genes are active. The use of RNA probes, especially antisense RNA probes, has become a preferred approach in situ hybridization due to their high sensitivity and specificity for target RNA sequences. This method is widely used to study gene expression in developmental biology, disease pathology, and other areas where understanding the localization of gene activity is crucial.

Stage 1 - ​Sample storage

Proper storage of tissue samples is critical for preserving nucleic acids and ensuring reliable results in situ hybridization experiments. To prevent RNA degradation, tissue samples should be handled with care and stored under conditions that inhibit RNase activity. Common approaches include flash-freezing samples in liquid nitrogen immediately after collection or fixing them in formalin followed by paraffin embedding (formalin fixed paraffin embedded, FFPE). FFPE tissues are particularly valuable for ISH, as they can be stored for long periods without significant loss of RNA integrity. Regardless of the method, it is important to keep tissue samples in a cool, dry environment and avoid repeated freeze-thaw cycles to maintain the quality of nucleic acids for downstream analysis.

Tissue preparation and sectioning

The preparation and sectioning of tissue samples are essential steps in the in situ hybridization protocol. Proper fixation, using agents such as paraformaldehyde or formalin, preserves the structure of the tissue and the integrity of nucleic acids, making them accessible for probe hybridization. After fixation, tissues are typically embedded in paraffin to facilitate thin sectioning with a microtome, producing tissue sections that can be mounted on slides for ISH analysis. For certain applications, such as studying gene expression in zebrafish embryos, whole mount ISH is used, allowing researchers to analyze entire tissues or organisms without sectioning. Careful tissue preparation ensures that the target nucleic acid sequences remain intact and available for specific hybridization with RNA or DNA probes.

Preserving RNA is made difficult due to presence of RNase enzyme. This enzyme is found on glassware, in reagents and on operators and their clothing. RNase quickly destroys any RNA in the cell or the RNA probe itself so users must ensure they use sterile techniques, gloves, and solutions to prevent RNase contamination of the probe or tissue RNA. Proper storage and handling of any biological sample is essential to prevent RNA degradation and ensure reliable in situ hybridization results.

For best results on older slides, do not store slides dry at room temperature. Instead, store in 100% ethanol at -20°C, or in a plastic box covered in saran wrap at -20°C or -80°C. Such storage preserves slides for several years.

Stage 2 - Choice of probe

Selecting the appropriate probe is a key factor in the success of in situ hybridization experiments. RNA probes, such as digoxigenin-labeled RNA probes, are commonly generated by in vitro transcription from a DNA template and are designed to hybridize specifically to target RNA sequences within tissue samples. The specificity and sensitivity of the assay depend on the design of the probe, including its length and sequence complementarity to the target. Probes can be tailored to detect specific DNA or RNA sequences, enabling the study of particular genes or gene families. The choice of probe should be guided by the experimental goals, such as detecting gene expression changes or mapping the localization of specific transcripts in tissue sections.

RNA probes should be 250–1,500 bases in length; probes of approximately 800 bases long exhibit the highest sensitivity and specificity. Transcription templates should allow for transcription of both probe (antisense strand) and negative control (sense strand) RNAs. Clone into a vector with opposable promoters to achieve this. Circular templates must be linearized before making a probe, though PCR templates can also be used, a PCR amplified sequence can serve as a template for probe synthesis. An antisense RNA probe is typically synthesized to hybridize to the target mRNA.

DNA probes provide high sensitivity for in situ hybridization. They do not hybridize as strongly to target mRNA molecules compared to RNA probes, so formaldehyde should not be used in the post hybridization washes.

Probe specificity is important. If the exact nucleotide sequence of the mRNA or DNA in the cell is known, a precise complementary probe can be designed. If >5% of base pairs are not complementary, the probe will only loosely hybridize to the target sequence. This means the probe is more likely to be washed away during wash and detection steps and may not be correctly detected.

Stage 3 - Digoxigenin (DIG)-labeled RNA probe in situ hybridization protocol

This protocol describes the use of DIG-labeled single-stranded RNA probes to detect expression of the gene of interest in paraffin-embedded sections. Paraffin-embedded or formaldehyde-fixed samples are referred to as fixed tissue. Standardized methods, such as those published in Nature Protocols, provide reliable guidance for these procedures.

Deparaffinization

For frozen sections, start at Step 2. If using formaldehyde-fixed paraffin-embedded sections, continue with this step. Before proceeding, slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section. Place the slides in a rack and perform the following washes:

• Xylene: 2x3 min
• Xylene 1:1 with 100% ethanol: 3 min
• 100% ethanol: 2x3 min
• 95% ethanol: 3 min
• 70% ethanol: 3 min
• 50% ethanol: 3 min
• Rinse with cold tap water

Antigen retrieval

Digest with 20 µg/mL proteinase K in pre-warmed 50 mM Tris for 10–20 min at 37°C. Incubation time and proteinase K concentration may require optimization.

Rinse slides 5x in distilled water.

Immerse slides in ice-cold 20% (v/v) acetic acid for 20 sec. This permeabilizes the cells to allow access to the probe and the antibody.

Dehydrate the slides by washing for approximately 1 min per wash in 70% ethanol, 95% ethanol and 100% ethanol, then air dry.

Add 100 µL of hybridization solution to each slide.

Salt solution (20x)

• 4 M NaCl
• 100 mM EDTA
• 200 mM Tris-HCl pH 7.5
• 100 mM NaH₂PO₄.2H₂O

Denhardt's solution

• 10 g Ficoli
• 10 g PVP (polyvinylpyrrolidine)
• 10 g BSA (bovine serum albumin)
• 500 mL sterile dH₂O

Incubate the slides for 1 h in a humidified hybridization chamber at the desired hybridization temperature. Typical hybridization temperatures range between 5562°C.

Dilute the probes in hybridization solution in PCR tubes. Heat at 95°C for 2 min in a PCR block to denature the RNA or DNA probe. Chill on ice immediately to prevent reannealing.

Drain off the hybridization solution. Add 50–100 μL of diluted probe per section, covering the entire sample. Incubate in the humidified hybridization chamber at 65°C overnight. Cover the sample with a cover slip to prevent evaporation.

Stringency washes

Solution parameters such as temperature, salt and detergent concentration can be manipulated to remove non-specific interactions.

To prepare 1 L of 20x saline sodium citrate (SSC)

• 175.3 g NaCl (3 M)
• 88.2 g sodium citrate
• 800 mL sterile dH₂O
• Adjust to pH 5 using citric acid, fill to 1 L and autoclave

Wash

• 50% formamide in 2x SSC
• 3x5 min, 37-45°C

Wash away excess probe and hybridization buffer with higher temperatures (up to 65°C) for short periods of time. If left too long this can wash off too much of the hybridized probe RNA/DNA.

Wash 2

• 0.1-2x SSC
• 3x5 min, 25-75°C

This step removes non-specific and/or repetitive DNA/RNA hybridization.

Wash twice in MABT (maleic acid buffer containing Tween 20) for 30 min at room temperature. MABT is gentler than PBS and is more suitable for nucleic acid detection.

To prepare 5x MABT stock

• 58 g maleic acid
• 43.5 g NaCl
• 55 g Tween-20
• 900 mL water

Bring the pH to 7.5 by adding Tris base. About 100 g will be required. Bring final volume to 1 L.

Dry the slides

Transfer to a humidified chamber and add 200 µL blocking buffer to each section (MABT + 2% BSA, milk or serum). Block for 1–2 h at room temperature.

Drain off the blocking buffer. Add the anti-label antibody at the required dilution in blocking buffer. Check the antibody datasheet for a recommended concentration/dilution. Incubate for 1–2 h at room temperature.

Wash slides 5x10 min with MABT at room temperature.

Wash the slides 2x10 min each at room temperature with pre-staining buffer (100 mM Tris pH 9.5, 100 mM NaCl, 10 mM MgCl2).

For fluorescence detection, proceed to Step 18.

For other forms of detection, return the slides to the humidified chamber and follow the manufacturer’s instructions for color development.

Rinse slides in distilled water.

Air dry the slides for 30 min. Wash in 100% ethanol and air dry thoroughly.

Mount using DePeX mounting solution.

Applications of in situ hybridization

In situ hybridization (ISH) is a powerful molecular technique used to detect specific nucleic acid sequences within fixed cells and tissues. It plays a crucial role in visualizing gene expression, identifying the spatial distribution of DNA or RNA targets, and studying cellular processes in their native context. ISH is widely applied across a range of fields such as developmental biology, pathology, neuroscience, oncology, and infectious disease research. The technique supports both DNA and RNA detection, including small RNAs like microRNAs, and can be combined with other methods, such as immunostaining or fluorescence imaging, for multiplexed and high-resolution analysis. ISH is also valuable in clinical diagnostics, prenatal screening, and biomarker validation, offering precise localization of genetic material for both research and medical applications.