Principles of Fluorescence In Situ Hybridization

Using known nucleic acid molecules indirectly labeled with haptens (such as biotin or digoxigenin) or directly labeled with fluorophores as probes, the probe and target double-stranded DNA are denatured and then hybridized. Complementary heterologous single-stranded DNA molecules anneal under appropriate temperature and ionic strength conditions to form stable heterologous double-stranded DNA. The haptens are subsequently visualized via fluorophore-labeled avidin or anti-digoxigenin antibodies, and hybridization signals are observed under a fluorescence microscope.

Types of Probes for Fluorescence In Situ Hybridization

Sample Processing for Fluorescence In Situ Hybridization

1. Conventional Paraffin-Embedded Tissue Sections
Fresh tissue samples are processed through fixation, washing, dehydration, clearing, paraffin infiltration, embedding, sectioning, and baking to prepare conventional sections. Special attention should be paid to fixation (type of fixative, fixation time) and sectioning (slide selection, prevention of tissue detachment, section thickness).

(1) Fixation:
The conventional fixative is 4% neutral formaldehyde. For specimens (organs or tissues) requiring histopathological examination, they should be promptly immersed in an adequate volume of fixative after removal from the body. Delayed fixation can lead to cellular autolysis and DNA degradation, resulting in faint or eroded nuclear staining under DAPI and weak or absent hybridization signals. Over-fixation can cause excessive macromolecular cross-linking, making digestion difficult and leading to high background noise. Improper fixation significantly impacts subsequent result interpretation.

(2) Sectioning:
Sections are typically 3–5 μm thick. Thinner sections require shorter digestion times; over-digestion can cause significant signal loss and affect result interpretation. Thicker sections are harder to digest and may require extended digestion time, often leading to overlapping cells, unclear cellular boundaries, difficulty in individual cell counting, and high background noise, which can also compromise result interpretation.

2. Conventional Cytological Samples

(1) Hypotonic Treatment:
Hypotonic treatment uses osmotic pressure to expand cells and spread chromosomes, dispersing nucleolar material adhering to the chromosomes for clear observation of all chromosome structures on a single plane. Adequate hypotonic treatment is essential, but insufficient or excessive treatment should be avoided. Inadequate hypotonic treatment can significantly affect FISH results, though extending digestion time may partially mitigate the issue. However, severe over-hypotonic treatment often results in irreversible FISH outcomes.

(2) Pre-fixation:
After hypotonic treatment, the swollen cell nuclei become fragile. During pre-fixation, fixative should be added slowly and carefully. Carnoy’s fixative is commonly used for cell fixation, as it rapidly penetrates cells, preserves chromosome structure integrity, and is suitable for general tissue and cell applications.

(3) Dropping and Smearing:
High-quality cell smears should have an appropriate density and uniform distribution of cells. Overly dense smears lead to overlapping cells, while overly sparse smears contain insufficient cells, both of which hinder accurate diagnosis. If the sample exhibits severe cell aggregation, excessive cell debris, or impurities (which may cause autofluorescence and high background), further treatment with trypsin or collagenase before hypotonic treatment (commonly used for urine sediment cells or amniotic fluid cells) can improve cell distribution and produce cleaner background smears.

Advantages of Fluorescence In Situ Hybridization

1. Fluorescent reagents and probes are economical and safe.

2. Probes are stable and can be used for up to one year after labeling.

3. The procedure is time-efficient, yielding rapid results with high specificity and precise localization.

4. FISH can localize DNA sequences as short as 1 kb, with sensitivity comparable to radioactive probes.

5. Multicolor FISH enables simultaneous detection of multiple sequences by displaying different colors within the same nucleus.

6. FISH can be applied to both metaphase chromosomes on slides to detect numerical or structural changes and interphase chromosomes in suspension to analyze DNA structure.

   
   

KMD Bioscience provides comprehensive one-stop FISH technical services, ranging from probe design, sample processing, hybridization detection, and gene expression studies to data analysis. Our standardized and streamlined protocols enhance the accuracy of experimental results while minimizing false positives and false negatives, ensuring high-quality FISH services for our clients. Our economical and safe fluorescent reagents and probes enable rapid completion of experiments, helping clients reduce the complexity and time required for laboratory FISH procedures. This approach also lowers experimental costs, conserves precious samples, and provides robust support for our clients' scientific research endeavors.

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