Fluorescent In Situ Hybridization Principle, Steps. Technique and Applications

Fluorescent In Situ Hybridization

The fluorescent in situ hybridization (FISH) is also known as interphase cytogenetics. In this technique, the double-stranded DNA is at first converted into single-stranded DNA, and then subsequently a fluorescent-tagged probe is used to visualize the target DNA.

Advantages Of Fluorescent In Situ Hybridization 

Fluorescent In Situ Hybridization has many advantages over conventional cytogenetic techniques. It is possible to do a fluorescent in situ hybridization on the paraffin-embedded tissue material, and therefore it can be used in archival materials.

Fluorescent In Situ Hybridization bypass the tedious cell culture technique. Most importantly cytogenetic abnormality of the cells can be demonstrated along with the morphology of the cell, and these two can be correlated

• No need for cell culture.
• Fluorescent In Situ Hybridization can be done on paraffin cell block material. 
• Archival tissue can be used for fluorescent in situ hybridization.
• Morphology of the cell can be seen along with cytogenetic abnormalities.
• High resolution.
• The slide can be stored for a long time.
• Fluorescent tags are safe and simple.

Limitations Of Fluorescent In Situ Hybridization 

Fluorescent In Situ Hybridization has the following limitations:

• The fluorescent in situ hybridization can be used only in the case of known chromosomal abnormalities as we use only the specific probes.

• It is not suitable for a screening test as only known chromosomal probes are used, whereas, in cell culture or conventional technique, we may get a wide range of chromosomal abnormalities.

• Fluorescent In Situ Hybridization does not give any allele-specific information.

Applications Of Fluorescent In Situ Hybridization:

Gain and loss of chromosomes: Fluorescent In Situ Hybridization is helpful to detect total gain or loss of chromosomes such as trisomy 12 in chronic lymphocytic leukemia which can be detected by fluorescent in situ hybridization in cytology or histology samples.

Chromosomal rearrangements: Fluorescent In Situ Hybridization is helpful to identify typical chromosomal translocation such as t (11; 22) (q24; q12) in Ewing's / primitive neuroectodermal tumor.

Gene amplification: Gene amplification such as HER-2 in breast carcinoma can be detected by fluorescent in situ hybridization.

Gene deletion: Gene deletion such as 9p21 deletion in urothelial cell carcinoma can be detected by fluorescent in situ hybridization.

Disease monitoring: To assess the progression or regression of disease and also to identify the minimal residual disease.

The Principles of Fluorescent In Situ Hybridization 

The basic principles of fluorescent in situ hybridization are:

• To convert double-stranded DNA into single-stranded DNA.
• DNA probes tagged with fluorescent dyes are also made to be single-stranded.
• The florescent-tagged single-stranded DNA probes are allowed to bind with the corresponding single-stranded DNA.
• The hybridized probe-target DNA complexes are visualized by a fluorescence microscope.

Steps To Do Fluorescent In Situ Hybridization 

1. Fixation: The cytology slides are fixed with 4% paraformaldehyde for 10 min. Histology tissues are already fixed in 10% buffered formalin.

2. Deparaffinization of the histology section:

(a) Cut 5-μ-thick section.

(b) Deparaffinize the histology section by baking the slide overnight at 56 ° C.

(c) Keep the slide in xylene for 10 min: two changes.

(d) Dehydrate by treating in 70% and 100% ethyl alcohol twice for 5 min.

(e) Dry the smear on the hot plate.

3. Pretreatment: 20 μg / ml proteinase K for 5–15 min

Dehydrate the smear by dipping in 70%, 80%, and 95% ethyl alcohol and dry the smear.

• Treat the smear with proteinase K solution (20 μg / ml) for 15 min at room temperature.

• (Proteinase K solution preparation: add 32 μl of proteinase K solution (25 mg/ml proteinase K solution) in 40 ml 2 × SSC, pH 7.4.)

• Gently wash the slide in deionized water.

• Dehydrate the smear. Saline sodium citrate (SSC): 

Keep pH 7.2 by adding drops of 10 N solution of NaOH. Now add water and make it 1 liter.

4. Denaturation:

Denaturing of target DNA: Denature DNA of the target cells in the smear by treating the smear with denaturing solution for 2 min time at 72 ° C.

Denaturing solution: 70% formamide in 2 × SSC (add 10 ml of double-distilled water, 5 ml of 20 × SSC, 25 μl of 250 mM EDTA, and 35 ml of formamide).

Denaturing of probe DNA: Add 1 μl of the labeled probe with 9 μl of 65% formamide solution, 10% dextran sulfate in 2 × SSC.

Now heat the mixture at 75 ° C for 5–6 min.

5. Hybridization:

• Add 10 μl of denatured probe solution over the slide.

• Put a coverslip over the smear and close the margins of the coverslip.

• Incubate it at 37 ° C for 1 day (24 h).

6. Post-hybridization:

• After the incubation, remove the coverslip gently, and rinse the slides in SSC for 5 min twice.

• Put the slides in a Coplin jar filled with prewarmed SSC at 70 ° C.

• Keep the slide in a Coplin jar filled with SSC at room temperature.

7. Visualization:

If the probes are directly labeled with the fluorochrome dye, then no further procedure is needed. In that case, counterstain the slide by 5 μl DAPI / antifade solution. Now visualize the cells by an epifluorescence fluorescence microscope.

Different Types Of Fluorescent In Situ Hybridization 

1. Three-dimensional Fluorescent In Situ Hybridization (3D FISH):

In this type of fluorescent in situ hybridization, multiple images of the nuclei are taken, and with the help of suitable software, a three-dimensional image is made. 3D fluorescent in situ hybridization helps to study the topology of the genes with respect to the chromosomal territory within the nucleus.

2. Living cell cytogenetics (four-dimensional fluorescent in situ hybridization ):

A fluorescent-tagged nucleotide can be incorporated into DNA that may help in the simultaneous visualization of DNA distribution and genomic organization in the living cells.

3. Multi-colored Fluorescent In Situ Hybridization (M-FISH) / spectral karyotyping (SKY):

In the case of the SKY technique, DNA is tagged with different fluorochrome dyes (the chromosome-specific painting probes), and all the chromosomes are stained. The different chromosomes therefore takes different color. There are three essential steps of M-fluorescent in situ hybridization :

(a) Hybridization: The fluorescent-tagged whole chromosome probes (WCP) are hybridized with the metaphase chromosome spread. WCPs consist of multiple probes tagged with spectrally different fluorochrome in a combinatorial manner.

(b) Visualization and image acquisition: In this step, the images of the chromosomes are visualized by the fluorescence microscope with attached filters. Subsequently, the images are acquired by the digital camera and appropriate software.

(c) Analysis: Finally, the images are analyzed with the help of specialized software to find out any structural chromosomal abnormalities.