9 ISH Tips You Can't Ignore

In situ hybridization (ISH) is a potent and versatile technique for detection of specific nucleic acid sequences in cells or tissues. As with any experimental method, careful control of each step is required to obtain reliable and reproducible results. This short mini-review provides 9 succinct but essential tips for successful ISH. These include the most important considerations for sample fixation and RNA protection, probe design, hybridization conditions and signal detection. Important pitfalls are noted at each step, as well as crucial optimizations to maximize signal, minimize background and to improve reproducibility.

Sample Fixation: Timely and Appropriate

Tissue fixation is the most important first step in sample preparation for ISH experiments. The most commonly used fixatives are 4% paraformaldehyde (PFA) and 10% neutral buffered formalin (NBF). However, fixation time should be optimized based on the tissue type and section thickness:

• Cryosections (7-15 µm): Fix at room temperature for 15-30 min.
• Paraffin sections: Fix in 4% PFA for 6-48 h.

Temperature and buffer condition are also important. It is always recommended to fix in RNase-free PBS at room temperature or 4°C (pH ≈ 7.4). Fixation in acidic conditions will result in RNA degradation.

After fixation, wash the tissue with PBS several times. This will remove the residual fixative and avoid potential inhibitory effects on enzymatic activity and blocking probe penetration.

Avoid using fixatives with heavy metals (mercury, chromium, etc.) as they can react with nucleic acids and interfere with hybridization and signal detection.

Preventing RNA Degradation: Protection at Every Step

All buffers, distilled water, and laboratory materials used for ISH experiments should be RNase-free. Prepare buffers in DEPC-treated water or use commercially available RNase-free reagents.

After collection, transfer tissues into ice-cold RNase-free PBS within 30 min and then immediately fix or freeze:

• Short-term storage: Freeze at -20 °C.
• Long-term storage: Cryosections at -80 °C; paraffin sections can be stored in 100% ethanol at -20 °C.

Minimize exposure to air through the workflow. When it is not avoidable, add RNase inhibitors (e.g., RNasin).

Pretreatment: Controlled Permeabilization

To inhibit endogenous peroxidase activity and decrease background during DAB staining, incubate the sections in 3% H₂O₂ at room temperature for 10-15 min.

In the case of FFPE samples, antigen retrieval in citrate buffer (pH 6.0) at 95-100°C for 10-20 min is recommended to reverse formaldehyde crosslinks, enhancing probe accessibility.

Proteinase treatment duration must be optimized. Choose the appropriate proteinase K concentration (1-20 µg/mL) and incubate at room temperature for 5-30 min.

• Excessive digestion causes tissue loss and distortion.
• Insufficient digestion compromises probe penetration.

Optimization by pre-experiment is strongly recommended.

Probe Design: Targeting the Right Region

Probe design should target the coding region (CDS) or the 3' untranslated region (3' UTR) of mRNA with the latter being preferable because it offers better sequence specificity. Probe length should be in the range of 50-150 bp-shorter probes facilitate penetration, whereas longer probes result in stronger specificity. Avoid targeting repetitive elements (e.g., poly-A tails), and confirm probe specificity with NCBI BLAST to avoid cross-hybridization.

In the case of multiplex ISH, use spectrally distinct fluorophores (e.g., FITC, Cy3, Cy5) with emission peaks spaced by at least 50 nm to reduce signal overlap or crosstalk.

Probe Labeling and Concentration Optimization

Radioactive labeling (e.g., ³⁵S) is appropriate for low-abundance targets, but radiation safety precautions are necessary. Labeling efficiency should be ≥ 10 dpm/µg.

Non-radioactive labeling (e.g., DIG or biotin) is preferred for safety and convenience; labeling density is recommended at 2-5 label molecules per 100 bp (verified by dot blot assays).

A probe concentration gradient test spanning 0.1 to 5 µg/mL must be established before hybridization to determine the best working concentration.

• mRNA ISH: 0.5-2 µg/mL
• DNA ISH: 1-3 µg/mL

Excessive probe concentration increases background noise, while insufficient concentration weakens signal intensity.

Hybridization: Precise Temperature and Time Control

Hybridization temperature should be tailored to the probe's GC content:

• DNA probes (GC 40-60%): 37-42°C
• RNA probes: 45-55°C (under RNase-free conditions)

Too high a temperature causes probe dissociation. In contrast, too low a temperature causes increased nonspecific binding.

Hybridization times are:

• Short probes (< 100 bp): 4-8 h
• Long probes (> 150 bp) or low-abundance targets: 12-16 h (overnight at 4°C)

To prevent tissue drying and background accumulation, do not exceed 24 h.

Post-Hybridization Wash: Gradual and RNase-Safe

After hybridization, perform a series of washes with increasing stringency to remove unbound probes:

• 2 × SSC + 0.1% SDS at room temperature, 2 × 5 min;
• 0.1 × SSC at 60-65°C, 2 × 15-20 min.

For RNA ISH, add RNase inhibitors (e.g. 0.1 U/µL RNasin) to all washing steps. All solutions should be prepared in DEPC-treated water to protect the target RNA.

Signal Detection: Stepwise and Temperature-Controlled

Non-radioactive detection

• DIG-labeled probes: Incubate in anti-DIG-alkaline phosphatase (diluted 1:500-1:2000) for 1-2 h at RT. Apply substrate NBT/BCIP, which generates a blue-violet precipitate, for signal visualization (bright-field detection).
• Biotin-labeled probes: Incubate with streptavidin-HRP (1:1000-1:5000 dilution) and detect with DAB substrate, resulting in a brown precipitate. Adjust color development time (5-30 min) to avoid saturation of the signal.

Fluorescent detection

Use anti-fade mounting medium which may contain DAPI for staining nuclei and protect the sample from light exposure during detection and afterward to prevent photobleaching.

Background Control and Result Validation

Use proper controls to verify the specificity of the ISH results:

• Blank control: no probe added.
• Negative probe control: The probe sequence exhibits no similarity to the target sequence.
• RNase-treated control (RNA ISH only): verify that the signal comes from RNA.

For quantification:

• Bright-field ISH: Analyze the ratio of positive cells or signal intensity (IOD) using the ImageJ software.
• Fluorescent ISH: Measure the mean fluorescence intensity (MFI) using confocal microscopy.

Measure at least three random fields (≥ 100 cells per sample) to avoid statistical bias.

Creative Bioarray Relevant Recommendations