Chromogenic in situ hybridization (CISH) is a
histological technique used to detect specific
nucleic acid sequences within the tissues. It combines traditional in situ hybridization with immunohistochemistry to visualize the presence and location of DNA or RNA sequences in tissue samples using chromogenic dyes. This makes it a valuable tool in
pathology and diagnostic
molecular biology.
CISH involves the use of labeled
probes that specifically bind to target nucleic acid sequences in the tissue. These probes are often labeled with
digoxigenin or biotin, which can then be detected using antibodies conjugated with enzymes such as
horseradish peroxidase (HRP) or alkaline phosphatase. The enzyme catalyzes a colorimetric reaction that produces a visible chromogenic signal, indicating the presence of the target sequence.
CISH is widely used in
cancer research and diagnosis to detect gene amplifications, deletions, and translocations. It is particularly valuable in identifying
HER2 amplification in breast cancer, which is crucial for determining the appropriate therapeutic regimen. Additionally, CISH is used to detect viral infections (such as
HPV) and to study the
gene expression patterns in various diseases.
One of the primary advantages of CISH is that it allows for the
visualization of target sequences within the context of tissue morphology, which is preserved during the procedure. This localization is particularly important for understanding the spatial distribution of genetic alterations within heterogeneous tissues. Unlike
FISH, CISH does not require a fluorescence microscope, as the signals are visible under a standard bright-field microscope. This makes CISH more accessible and easier to interpret for many laboratories.
Despite its benefits, CISH has some limitations. The sensitivity of CISH can sometimes be lower compared to FISH, and the detection of some low-abundance targets might be challenging. Additionally, the procedure can be time-consuming and requires careful optimization of protocol parameters such as probe concentration, hybridization conditions, and enzymatic detection steps to achieve reliable results.
While both CISH and FISH are in situ hybridization techniques, they differ primarily in the detection methods. FISH uses fluorescently labeled probes and requires a fluorescence microscope for signal detection. In contrast, CISH uses chromogenic dyes, allowing visualization with a standard bright-field microscope. FISH is generally more sensitive and can detect low-abundance targets more effectively, but CISH offers the advantage of easier interpretation and does not require specialized equipment.
The CISH procedure typically involves the following steps:
Sample preparation: Tissue sections are prepared and fixed on slides.
Pre-treatment: The tissue is treated to permeabilize cell membranes and remove proteins that may interfere with hybridization.
Hybridization: Labeled probes are applied to the tissue and allowed to hybridize with the target sequences.
Washing: Excess probes are washed away to reduce background signals.
Detection: Enzyme-conjugated antibodies are applied to bind to the labeled probes.
Color development: A chromogenic substrate is added, producing a visible signal where the target sequence is present.
Analysis: The stained tissue sections are examined under a bright-field microscope.
Conclusion
CISH is a powerful histological technique that bridges the gap between molecular and morphological analysis. Its ability to provide spatial context to genetic alterations makes it indispensable in clinical diagnostics and research. Despite some limitations, CISH remains a valuable tool for understanding the complexity of tissue biology and disease.
