Fluorescence In Situ Hybridization Protocol and FAQs

Fluorescence in situ hybridization ( FISH ) is a molecular biology technique used to detect and localize the location of specific nucleic acid sequences ( such as DNA or RNA ) in cells or tissue sections. This technology is widely used in genomics, cytology and pathology research, especially in gene localization, chromosome abnormality analysis and gene expression detection.

The Principle of FISH

Fluorescence-labeled nucleic acid probes are used to hybridize with target DNA or RNA sequences in cell or tissue samples to locate and detect specific nucleic acid sequences at the molecular level. FISH is a very sensitive and highly specific technique that can accurately identify and display the location of the target gene or RNA in cells or tissue sections.

The Steps of FISH ( fluorescence in situ hybridization ) Experiment

Sample Preparation

Before FISH experiments begin, samples, usually cell or tissue sections, need to be prepared.

Cell or tissue preparation

Select the appropriate cell or tissue samples, common sources include blood, tissue sections, cell culture, etc. The sample needs to maintain the original spatial structure in order to accurately locate the target sequence.

Fixation

Using chemical reagents (such as formaldehyde, glacial acetic acid, etc.) to fix the sample. The purpose of immobilization is to keep the cells or tissues in the sample in the original structural state to prevent the degradation of cells or nucleic acids. The commonly used fixation methods are : 4 % formaldehyde fixation, glacial acetic acid fixation, etc.

Penetration

After the sample is fixed, in order to allow the probe to penetrate into the cell or tissue, penetration is usually required. Commonly used permeabilizers include Triton X-100, Saponin, etc. These reagents can destroy the cell membrane and allow nucleic acid probes to enter the cell.

Preheating treatment (optional)

In some cases, the sample may need to undergo thermal denaturation treatment to unzip the DNA double strands, which facilitates the binding of the probe to the target sequence.

Probe Design and Labeling

Probe design

FISH probes are usually short single-stranded nucleic acids (DNA or RNA) that target DNA or RNA sequences. These probes must have a high degree of specificity and can be complementary to the target sequence. According to the needs of the experiment, the design of the probe needs to consider the specificity, length and stability of the target gene or sequence.

Probe labeling

In order to visualize the probe in the experiment, the probe is usually labeled with a fluorescent dye. The commonly used fluorescent labeling dyes are FITC (green), Cy3 (red), Alexa Fluor series, etc. The labeling of the probe can be either direct labeling (connecting fluorescent molecules at the end of the probe) or indirect labeling (using biotin or other markers, and then using secondary antibody labeling).

Hybridization

Hybridization solution preparation

The labeled probe was mixed with a hybridization buffer, usually using a buffer containing an appropriate concentration of salt and organic solvent, such as SSC (salt-sodium citrate buffer) or Hybe buffer. Ensure that the temperature, salt concentration and pH value of the hybridization solution are suitable for the binding of the probe to the target sequence.

Probe addition

The labeled probe solution was added to the sample (such as tissue sections or cells). At this time, the probe will hybridize with the target DNA or RNA sequence, and the hybridization is usually carried out at higher temperatures (such as 37 ° C to 55 ° C). The specific temperature is adjusted according to the GC content of the probe and the characteristics of the target sequence. Usually, the hybridization time is a few hours to overnight.

Washing

Remove non-specific binding

After the hybridization reaction is completed, the sample needs to be washed to remove the probe without specific binding and reduce the background signal. The washing process usually includes washing with low-salt buffer (such as 2 × SSC), and the temperature can be set to room temperature or slightly higher to remove non-specifically bound probes.

Enhanced signal

In some cases, enhancement steps can also be used, such as the use of antibody-labeled secondary antibodies or fluorescence enhancement reagents, to further improve the intensity and specificity of the signal.

Signal Detection and Observation

Fluorescence microscope observation

After washing, the sample can be observed by fluorescence microscope when it is ready. The microscope uses an appropriate filter to excite the fluorescent dye labeled on the probe and capture the emitted fluorescence signal. Different fluorescent dyes have different emission wavelengths, so multiple target sequences can be detected simultaneously by selecting different filter combinations.

Photographing and analysis

The fluorescence signal under the microscope shows the location of the target DNA or RNA sequence. The experimental data can be saved by photographing the image, and the location, number or expression pattern of the target sequence can be further analyzed.

Postprocessing and Data Analysis

Image analysis

Quantitative analysis of fluorescence images was performed using image analysis software (such as ImageJ, Metamorph, etc.) to evaluate signal strength, signal location, etc. This is very important for gene localization, gene expression analysis and multiple signal localization in multiple FISH experiments.

Result explanation

According to the location and intensity of the fluorescence signal, the distribution of the target gene or RNA in the cell or tissue was analyzed. For example, analyze the chromosomal location of genes, the spatial distribution of gene expression, or the genetic differences between different samples.

Figure 1. The Schematic diagram of the entire cardioFISH process. (Reference source: Fluorescence in situ hybridization protocol for cardiomyocytes.）

Typical FISH Probe Types

DNA Probe

Used to detect DNA sequences, usually used for chromosome localization analysis. The number, structure and gene localization of chromosomes can be analyzed by DNA probes.

RNA Probe

Used to detect specific RNA molecules, usually for gene expression analysis. Through RNA probes, the expression levels of specific genes can be localized and quantified in cells or tissues.

Multiplex FISH Probe

Multiple target sequences can be detected simultaneously by using fluorescently labeled probes of different colors. This multiple probe application can effectively improve the efficiency of the experiment and help researchers to observe the localization information of multiple genes or RNA at the same time.

The Specificity and Sensitivity of FISH Technique

Specificity

The specificity of FISH comes from the complementarity between the probe and the target sequence. Hybridization can be successful only when the probe is completely matched with the target sequence. The design of probes needs to be selected according to the uniqueness and redundancy of the target sequence to avoid non-specific hybridization.

Sensitivity

FISH can detect low-abundance target sequences because the labeled probe can efficiently bind to the target sequence and can detect extremely weak signals through the amplification of the fluorescence signal. This allows FISH to be used not only for large-scale genomic analysis, but also for gene localization and expression detection at a single cell or subcellular level.

Alpha Lifetech has many years of project experience, which can better serve the various needs of customers. Alpha Lifetech provides fluorescence in situ hybridization (FISH) detection services for the detection and localization of specific DNA or RNA sequences in cell or tissue samples. FISH uses fluorescently labeled probes that bind to complementary sequences to allow specific gene, chromosome or RNA markers to be visualized under a fluorescence microscope.

FAQ

1. In FISH experiments, the fluorescence signal may be affected by non-specific binding, resulting in too strong background signal and affecting the accuracy of the results.

(1). Strictly control the hybridization conditions : ensure that the temperature, salt concentration, pH value and other conditions of the hybridization solution meet the binding requirements of the probe and the target sequence. Too low hybridization temperature may cause the probe to bind to the non-target sequence, thereby increasing the background signal. (2). Optimize washing steps : Use appropriate washing conditions (such as higher salt concentration of SSC buffer) to remove non-specific binding probes and reduce background signals. The temperature during washing is usually set at room temperature or slightly higher to effectively remove the unbound probe. (3). Selectively increase the washing time : prolong the washing time and increase the number of washings to ensure the removal of non-specific binding probes. (4). Antibody blocking : The sample is blocked with blocking reagents (such as BSA or protamine) to reduce non-specific binding.
2. The fluorescence signal is weak or the signal cannot be observed, resulting in unclear experimental results.

(1) Increasing the probe concentration : If the signal is too weak, the concentration of the probe can be increased or the hybridization time can be prolonged, so that the probe can bind to the target sequence more fully. (2) Increase the fluorescence intensity of fluorescent dyes : Select stronger fluorescent dyes (such as Alexa Fluor series, Cy5, etc.), or use secondary antibodies to enhance the signal. (3) Optimization of hybridization conditions : The conditions such as salt concentration and pH value of the hybridization solution were optimized to ensure that the binding of the probe to the target sequence was more efficient. (4) Increase the amount of labeled probes : For multiple FISH experiments, if the signal is weak, you can consider increasing the amount of different fluorescent probes to enhance the signal intensity of each target.
3. The probe non-specifically binds to the non-target sequence, resulting in inaccurate results.

(1) Increasing the hybridization temperature : Increasing the hybridization reaction temperature can increase the specific binding of the probe to the target sequence and reduce the non-specific binding. The choice of temperature depends on the GC content of the probe. Usually, the temperature should be optimized according to the annealing temperature (Tm value) of the probe. (2) Optimize probe design : Ensure that the sequence of the probe design is highly specific and avoid complementary pairing with other non-target sequences. (3) Use appropriate washing conditions : increase the strictness of the washing steps, and use a higher concentration of salt buffer (such as 2 × SSC) to remove non-specifically bound probes.
4. The probe failed to enter the cells or tissues effectively, resulting in the failure of the experiment.

(1) Permeable cells : The samples were treated with appropriate permeabilizers (such as Triton X-100, Saponin, etc.) to ensure that the probe could effectively penetrate the cell membrane. The time and concentration of permeation should be appropriate to avoid cell damage. (2) Optimization of permeabilization conditions : Different cell or tissue types may require different permeabilizers or permeabilization conditions to test different permeabilization methods to ensure the best probe entry effect.
5. The probe failed to hybridize with the target DNA or RNA sequence, resulting in the failure of the experiment.

(1) Optimized probe design : Ensure that the probe sequence is sufficiently complementary to the target sequence, and select the appropriate length (usually 20-50 base pairs) to improve the hybridization efficiency. (2) Adjust the hybridization conditions : adjust the hybridization temperature and salt concentration according to the GC content of the target sequence, so that the probe can effectively bind to the target sequence. For sequences with higher GC content, higher hybridization temperature is required. (3) Prolong hybridization time : Increase hybridization time, especially for low abundance target sequences, overnight hybridization can be considered to ensure sufficient binding.

reference

[1] Bayani J, Squire JA. Fluorescence in situ Hybridization (FISH). Curr Protoc Cell Biol. 2004;Chapter 22:. doi:10.1002/0471143030.cb2204s23

[2] Chrzanowska NM, Kowalewski J, Lewandowska MA. Use of Fluorescence In Situ Hybridization (FISH) in Diagnosis and Tailored Therapies in Solid Tumors. Molecules. 2020;25(8):1864. Published 2020 Apr 17. doi:10.3390/molecules25081864

[3] Gozzetti A, Le Beau MM. Fluorescence in situ hybridization: uses and limitations. Semin Hematol. 2000;37(4):320-333. doi:10.1016/s0037-1963(00)90013-1

[4] Jiang J. Fluorescence in situ hybridization in plants: recent developments and future applications. Chromosome Res. 2019;27(3):153-165. doi:10.1007/s10577-019-09607-z

[5] Yao Z, Bai L, Nie Y. Fluorescence in situ hybridization protocol for cardiomyocytes. J Mol Cell Cardiol. Published online February 13, 2025. doi:10.1016/j.yjmcc.2025.02.003