Pathology Detection-Fluorescence in Situ Hybridization Cautions and FAQs

What’s fluorescence in situ hybridization?

Fluorescence in situ hybridization (FISH) is based on population-specific DNA sequences at different taxonomic levels of known microorganisms, using fluorescence-labeled specific oligonucleotide fragments as probes and hybridize with DNA in the environmental genome to detect the presence and abundance of the specific microbial population.

Background and advantages of fluorescence in situ hybridization

Fluorescence in situ hybridization came out in the late 1970s. It has been widely used in the chromosomal abnormalities study. Recent years, with the increasing probes number used in FISH, especially the emergence of full Cosmid probe and chromosome in situ suppression hybridization technology, FISH technology is widely used not only in cytogenetics, but also in oncology research, such as gene diagnosis and gene mapping.

There are many disadvantages in the original radioisotope in situ hybridization technique, such as relabeling the probes in each test, obvious instability in the labeled probes, long exposure time need and environmental pollution, etc.

In addition, errors in counting may be caused due to the different planes of radioactive silver particles and chromosome aggregation. Compare with that, FISH has advantages as follows:

1.Easy to operate. Stable after labeling. It can be used for two years after marking.

2.Sensitive. Get the results quickly.

3.Several different probes can be tested in one sample simultaneously.

4.It can be used not only in the study of chromosome number or structure changes in dividing cells, but also in the study of chromosome number and genetic alteration of cells in intermitosis.

Fluorescence in situ hybridization is an important non-radioactive in situ hybridization technique. The basic principle is: If the target DNA on the detected chromosome or DNA fiber slice is homologously complement to the nucleic acid probe, a hybrid of target DNA and nucleic acid probe would be formed after denaturalization-annealing-refolding.

Label a certain nucleotide of a nucleic acid probe with a reporter molecule such as biotin or digoxin, utilize the immunochemical reaction between the reporter molecule and a fluorescein-labeled specific avidin to qualitative, quantitative and relative localize the tested DNA with the fluorescence detection system.

Even so, the operation of this technology is relatively complicated. Here are the summary of the cautions and FAQs of this operation.

1. Experimental conditions and personnel training for FISH operation

Apart from a dark room to operate fluorescence experiment and microscopic observation, in situ hybridization machine, fluorescence microscope, microscope filter, water bath, vortex mixer, centrifuge, pipette, FISH probe kit, anhydrous ethanol, xylene, deionized water, rubber cement, transparent nail polish and other equipment and reagents are also needed.

Because of the sensitivity and objectivity of FISH detection, any small mistake may cause error in the interpretation of the results, then affects the rational use of drugs in clinical diagnosis. For this reason, excellent and standard skills of the researchers are needed.

Each pathology department conducting molecular pathological examination is recommended to set up a special molecular pathology room and be operated by a special person. This person should master relevant molecular biology principles, have been trained specially and have certain laboratory work experience.

2. The choice and time of fixed solution

Paraffin tissues are recommended to be fixed with 10% neutral buffered formalin for 6 to 48 hours. Other fixed solution may have certain effects on the experimental results.

The isolated tissue should be fixed as soon as possible (within 30 minutes). If the sample is not fixed in time, the fluorescence signal will be weakened.

The volume of the fixed solution is 4 to 20 times that of the tissue, otherwise the fixation is insufficient, lead to little or no signal.

Moreover, in order to ensure the accuracy of the signal, the wax block is best to be tested within 2 years, and the cut slice is best to be tested within 6 weeks.

3. Selection of tissue sections and slides

Recommend to cut tissue sections 4 to 5 µm thick because either too thick or too thin slices will impact the strength of the signal. Slices should be intact, good quality, otherwise it will drop down during pretreatment.

Recommend to use anti-off slides, and better cation or anion-specific slides could be used in some laboratories to prevent dropping down effectively.

4. Slice pretreatment

The pretreatment includes boiling and protease digestion. It is easy to fall off during the boiling process if the tissue is not properly processed, the slice is too thick or it is baked insufficiently. Recommend to bake the slice at 56°C overnight.

Control the temperature and time strictly during boiling. Enzyme digestion is one of the key steps in FISH. If you are unfamiliar with the tissue, you can try the recommended reaction time firstly, and then re-stain DAPI under the fluorescence microscope. Neither hole(digest excessively) nor mist (digest deficiently) exists under the DAPI channel is best. No obvious background under the red and green channel is better. If the background is dark, extend the digestion time appropriately.

In addition, extend the digestion time appropriately for old wax tissue sections, otherwise a large amount of autofluorescence will appear. Little or no signal will appear if the digestion is  insufficient, and the signal is better when digest sufficiently.

5. Denaturation and hybridization

Denaturation and hybridization are the most critical steps in this experiment. The time and temperature of denaturation is the key to the success of hybridization. To ensure the stability of temperature during denaturation, it is recommended to use a professional in situ hybridization instrument to conduct denaturation and hybridization. It can not only reduce the experimental steps, but also good for controlling experimental conditions such as time, temperature and store in dark place. In order to avoid loss of the hybridization solution during denaturation and hybridization and to prevent the film from drying it is necessary to seal with rubber cement around the cover glass.

6. Washing temperature, time and cover preservation after hybridization

Washing after hybridization is very important for the whole experimental results. Firstly, it should be ensured that the hybridization washing solution is properly prepared. A high temperature of the washing solution or a long time washing may cause a weak signal. A low temperature of the washing solution or a short time washing may cause disordered signals, poor contrast between red and green signals or low specificity.

After re-staining DAPI, cover the glass fix the four corners of the glass with nail polish to prevent the glass from falling off under the oil immersion lens. Since the fluorescent signal is easily quenched, it should be observed immediately after staining. If cannot, place the slice in a box and stored at -20°C within 2 weeks.

7.Set a control

A standard control is required in each experiment: Use standardized control materials (positive, negative, and suspicious) in each round of testing. Results couldn’t be determined if the control material didn’t show an expected result.

8. Determine the results

Select cells with uniform size of nucleus, intact nuclear boundaries, uniform DAPI staining, no overlapping nucleus and clear signals. Randomly count two-color signals in at least 20 invasive cancer cell nucleus. For cases with uncertain FISH results, the signals in 20 nuclei need to be recalculated or counted by another pathologist.

If it is still a threshold value, immunohistochemical detection should be performed (if it is not performed before FISH detection) or select different tissue blocks to retest. A standard test and a correct results interpretation are important for choosing the right patients and instruct clinical treatment.