In Situ Hybridization (ISH) Protocol
Licia Miller Product Manager
In situ hybridization (ISH) is an experimental technique used to detect and locate specific nucleic acid sequences (DNA or RNA) in cells or tissues. It uses labeled nucleic acid probes to specifically bind to target nucleic acid sequences, thereby observing and analyzing the expression and distribution of nucleic acids in the natural location of cells or tissues.
The basic principle of in situ hybridization is to use the specific binding between complementary nucleic acid sequences. In the experiment, synthetic labeled probes (usually RNA or DNA) are applied to fixed cells or tissue sections, and the probes hybridize with the target nucleic acid sequences to form a double-stranded structure. Through subsequent washing and detection steps, these hybridization signals can be identified and visualized to determine the location and expression level of the target nucleic acid in the sample.
In in situ hybridization (ISH) experiments, the probe labeling and detection system is the key to nucleic acid localization and expression analysis. Labeling and detection systems based on digoxigenin, biotin, and fluorescent labeled molecules are common in situ hybridization detection methods.
Among them, the digoxigenin (DIG) labeling system is a widely used labeling method that uses digoxigenin antibodies for detection. Digoxigenin has high specificity and can effectively bind to the probe, and its antibodies do not cross-react with other biological molecules. The advantage of this system is its high signal-to-noise ratio and sensitivity, which is suitable for the detection of low-copy RNA. However, the detection process of digoxigenin is relatively complicated and usually requires immunoassay and substrate reaction.
This experimental protocol mainly introduces the digoxigenin (DIG)-labeled RNA single-stranded in situ hybridization protocol to detect the expression of target genes in paraffin-embedded sections.
Ⅰ.Sample preservation
General sample storage of frozen sections: store them in 100% ethanol at -20℃ , or store them in a plastic box covered with plastic wrap at -20℃ or -80℃. This storage method can keep the slides for several years. Be careful not to store the slides dry at room temperature.
RNA preservation: RNase enzymes are widely present in glassware, reagents, and operators' bodies and clothes. Therefore, it is necessary to pay attention to the sterility of the entire experimental process, gloves and solutions to prevent the probe or tissue RNA from being contaminated by RNase.
Ⅱ.Probe Selection
In in situ hybridization (ISH) experiments, the selection of appropriate probes is crucial to the success of the experiment. The selection of probes mainly involves DNA probes, RNA probes and oligonucleotide probes, which can be labeled by different enzymatic molecular reactions to facilitate the detection of specific nucleic acid sequences.
Oligonucleotide probes are a type of probe with a shorter length that usually avoids the problem of internal annealing of the probe and has better penetration during hybridization. The advantage of this type of probe is that they can more easily access the target, which is one of the key factors affecting the success of in situ hybridization. The shorter length of oligonucleotide probes allows them to diffuse more rapidly in the tissue, thereby improving the efficiency of hybridization.
RNA probes usually have higher detection specificity and sensitivity than DNA probes due to their single-stranded structure, high molecular binding force, and adaptability to high-temperature hybridization. The length of RNA probes is about 250-1500 bases, of which probes of about 800 bases are considered to have the best sensitivity and specificity. The preparation of RNA probes involves the construction of a transcription template that should support the transcription of the probe (antisense strand) and negative control (sense strand) RNA. The synthesized RNA probe needs to be verified for its sensitivity and specificity to the target fragment to ensure the accuracy of the experimental results.
DNA probes provide higher sensitivity for in situ hybridization, but compared with RNA probes, the hybridization strength between DNA probes and target mRNA molecules is weaker, so formaldehyde should be avoided during the washing process after hybridization to avoid destroying the DNA-RNA hybrid double strand. When synthesizing DNA probes, the length of the probe fragment needs to be controlled, usually around 300-1000bp, so that such a length can cover longer fragments of the target nucleic acid sequence, thereby increasing the sensitivity of the detection.
Probe specificity is critical. If the exact nucleotide sequence of the mRNA or DNA in the cell is known, highly accurate complementary probes can be designed based on this. If more than 5% of the base pairs are not complementary, the probe will only hybridize poorly to the target sequence. This means that the probe is more likely to be washed away during the washing and detection steps and may not be detected correctly.
Ⅲ.Experimental steps
1. Sample processing - dewaxing (this step can be skipped for frozen sections)
For paraffin-embedded sections fixed with formaldehyde , dewaxing and rehydration are first required . This step is very critical. If the paraffin is not completely removed, the staining effect of the sections will be poor.
Place the slides in the rack and perform the following washes:
Wash twice with xylene, 3 minutes each time; 1:1 mixture of xylene and 100% ethanol for 3 minutes; twice with 100% ethanol for 3 minutes each time; 95% ethanol for 3 minutes; 70% ethanol for 3 minutes; 50% ethanol for 3 minutes.
Wash with ice-cold tap water and place the slide in tap water. During subsequent operations, remember not to let the slide dry out, otherwise it will cause nonspecific antibody binding and high background staining.
2. Antigen Retrieval
2.1 Preheat 50 mM Tris containing proteinase K and incubate at 37°C. The incubation time is generally 10-20 minutes . The recommended concentration of proteinase K is 20 µg/mL.
In different experiments, the optimal enzyme concentration and incubation time will vary due to differences in tissue type, size, and fixation time. Insufficient enzymatic hydrolysis will lead to reduced hybridization signals, while excessive enzymatic hydrolysis will affect tissue morphology and the location of hybridization signals. It is recommended to determine the optimal conditions through proteinase K titration experiments.
2.2 Wash the slide 5 times with distilled water.
2.3 Immerse the slide in pre-cooled 20% (v/v) acetic acid for 20 seconds. This will permeabilize the cells and allow the probe or antibody to penetrate.
2.4 Wash the slides with 70% ethanol, 95% ethanol and 100% ethanol respectively (about 1 minute each time) to dehydrate them, and then air dry them.
3. Probe Preparation
The preparation of DNA probes includes the initial separation and purification of DNA fragments (or cloning of cDNA) and enzymatic probe labeling, and optional random primer labeling and nick translation methods. The preparation of RNA probes includes transcription vector cloning and transcription probe labeling. The preparation of oligonucleotide probes requires obtaining synthetic oligonucleotide fragments first, and then end labeling or tailing probe labeling. However, no matter what kind of labeled probe and labeling method is used, the probe labeling effect must be finally evaluated and the probe concentration must be accurately calculated.
4. Probe Hybridization
4.1 Prepare hybridization solution according to the following recipe and add 100 µL of hybridization solution to each slide.
Hybridization solution formula:
Reagents | Final concentration | Amount per 1mL of solution |
50% | 500 µL | |
Saline solution | 5 × | 250 µL |
5 × | 50 µL | |
Dextran sulfate | 10% | 100 µL |
Heparin | 20 U/µL | 10 µL |
SDS | 0.1% | 1 µL |
Saline solution (20 ×)
- 4 M NaCl
- 100 mM EDTA
- 200 mM Tris-HCl pH 7.5
- 100 mM NaH2PO4•2H2O
- 100 mM NaH2PO4•2H2O
- 5 g Polysucrose
- 5 g PVP (Polyvinylpyrrolidone)
- 5 g bovine serum albumin (BSA)
- 250 mL sterile dH2O
4.2 Place the slide in a humidified hybridization box at the desired hybridization temperature and incubate for 1 hour. The hybridization temperature usually ranges from 55 to 62°C.
4.3 Dilute the probe in hybridization solution in a PCR tube. Heat the RNA or DNA at 95°C for 2 minutes using a PCR instrument to denature it. Then immediately place the probe on ice to cool it down to prevent re-annealing.
4.4 Remove the hybridization solution. Add 50-100 μL of probe dilution to cover the entire sample. Incubate overnight at 65°C in a humidified hybridization chamber. Cover the sample with a coverslip to prevent evaporation.
In this step, RNA probes will hybridize with the corresponding mRNA, or DNA probes will hybridize with the corresponding cellular DNA. The optimal hybridization temperature of the probe depends on the percentage of bases in the target sequence. The ratio of cytosine to guanine in the sequence is a key factor.
5. Washing
Nonspecific interactions can be eliminated by adjusting solution parameters such as temperature, salt concentration, and detergent concentration.
5.1 Prepare 1 L of 20 × sodium citrate buffer (SSC)
Reagents | Required mass/volume |
Sodium chloride (3 M) | 175.3 g |
Sodium Citrate | 88.2 g |
ddH2O | 800 mL |
Adjust pH to 5 with citric acid, make up to 1 L and sterilize by autoclave.
5.2 First wash: Wash three times with 2× SSC containing 50% Formamide at 37-45°C for 5 minutes each time.
5.3 Second wash. Clear three times with 0.1 - 2 × SSC at 25-75°C for 5 minutes each. This step will eliminate non-specific and/or repetitive DNA/RNA hybridization.
Optimize the temperature and salt concentration for stringent washes as follows:
For very short DNA/RNA probes (0.5-3 kb) or very complex probes, wash temperatures should be lower (up to 45°C) and stringency less stringent (1-2 × SSC) .
For single loci or larger probes, the temperature should be around 65°C and the stringency should be high (less than 0.5 × SSC).
For repetitive probes, the temperature should be set to the highest setting and the stringency should be set to the highest setting.
5.4 Wash twice with MABT (maleic acid buffer containing Tween 20) at room temperature for 30 minutes each time. MABT is milder than PBS and is more suitable for nucleic acid detection.
Reagents | Required mass/volume for 5 × MABT stock solution |
Maleic acid | 58 g |
NaCl | 43.5 g |
Tween-20 | 55 g |
ddH2O | 900 mL |
Add Tris(hydroxymethyl)aminomethane to adjust pH to 7.5. Approximately 100 g of Tris(hydroxymethyl)aminomethane is needed. Make up to 1 L.
5.5 Dry the slide.
6. Blocking and Antibody Incubation
6.1 Transfer to a humidified chamber and add 200 µL of blocking buffer (MABT + 2% BSA, milk or serum) to each section. Block for 1 - 2 hours at room temperature.
6.2 Add anti-labeled antibody to blocking buffer at the required dilution and incubate at room temperature for 1 - 2 hours.
6.3 Wash the slides 5 times with MABT at room temperature for 10 minutes each time.
6.4 Wash the slides twice for 10 minutes each in pre-staining buffer (100 mM Tris pH 9.5, 100 mM NaCl, 10 mM MgCl2) at room temperature.
6.5 If fluorescence detection is required, skip to the next step. For other forms of detection, place the slide back into the humidified chamber and develop the color according to the manufacturer's instructions.
6.6 Wash the slides with distilled water.
6.7 Allow the slide to air dry for 30 minutes. Then wash with 100% ethanol and air dry thoroughly.
6.8 Use DePeX sealing solution to seal the slides, and then the test can be carried out.
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