Chromogenic in situ hybridization

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CISH workflow

Chromogenic in situ hybridization (CISH) is a laboratory technique used in the field of molecular biology and pathology to detect specific sequences of DNA or RNA within tissue sections. The method combines in situ hybridization, which involves hybridizing a complementary DNA or RNA probe to a specific nucleic acid sequence in the tissue, with chromogenic detection, where the hybridized probe is visualized using a chromogenic substrate. This allows for the localization and visualization of specific genetic material within the cells of a tissue section under a light microscope.

Overview

CISH is particularly useful in the diagnosis and research of cancer, where it can be employed to identify gene amplification, gene deletion, or chromosomal translocations. Unlike fluorescence in situ hybridization (FISH), which uses fluorescent probes, CISH uses enzyme-linked probes that produce a precipitate when exposed to a chromogenic substrate. This results in a colored precipitate at the site of the target sequence, which can be seen with standard light microscopy. This technique has the advantage of allowing the simultaneous observation of the tissue morphology and the specific genetic markers.

Procedure

The CISH procedure involves several key steps: 1. Tissue sections are prepared on slides and treated to allow probe penetration. 2. A labeled DNA or RNA probe is applied to the tissue section. The probe is designed to be complementary to the target sequence of interest. 3. The probe hybridizes to the target sequence within the tissue. 4. After hybridization, the slides are washed to remove any unbound probe. 5. A detection system is applied, which typically involves an enzyme-linked antibody that binds to the probe. The enzyme acts on a chromogenic substrate, resulting in a colored precipitate at the site of the probe-target hybridization. 6. The slides are then counterstained, mounted, and viewed under a light microscope.

Applications

CISH is widely used in clinical diagnostics and research. In oncology, it is used to detect and quantify gene amplification in tumors, such as HER2 amplification in breast cancer. This has important implications for prognosis and treatment, as HER2-positive cancers may be treated with targeted therapies. CISH is also used in the detection of viral infections in tissue sections, identification of chromosomal abnormalities, and the study of gene expression in specific tissues.

Advantages and Limitations

The main advantage of CISH over FISH is the ability to observe the chromogenic signal under a light microscope, allowing for the examination of the tissue morphology alongside the genetic information. This also facilitates the archival of slides, as the chromogenic signal does not fade over time like fluorescent signals. However, CISH may have lower sensitivity compared to FISH due to the chromogenic detection method, and multiplexing (detecting multiple targets simultaneously) is more limited with CISH.

Conclusion

Chromogenic in situ hybridization is a valuable tool in molecular pathology, offering a bridge between traditional histopathology and molecular genetics. Its application in cancer diagnostics and research continues to provide critical insights into the genetic alterations that drive cancer, aiding in the development of targeted therapies and improving patient outcomes.

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