Skip to main content

Molecular Biology

In Situ Hybridization (ISH)

In Situ Hybridization (ISH) is a technique that’s like a molecular GPS for the cell. Imagine you have methods like PCR or Southern blotting, which are fantastic, but they require you to first extract the nucleic acids from the cells. That’s like taking all the books out of a library, grinding them up, and then figuring out if a specific sentence exists somewhere in the pulp

In situ hybridization (ISH), on the other hand, lets us walk into the intact library, go to the right room (the cell), find the exact shelf (the chromosome), and put a glowing “sticky note” right on the book (the gene) we’re looking for. The term “in situ” is Latin for “in its original place,” and that’s the magic of this technique: we are detecting DNA or RNA sequences right inside the cell or tissue, preserving the all-important morphological context

The Principle: A Labeled Probe Finds Its Target

At its core, ISH is a hybridization assay. The main principle is the highly specific binding (hybridization) of a labeled, single-stranded nucleic acid probe to its complementary single-stranded target sequence within an intact cell or tissue section

  • Probe: A short, synthetically made piece of DNA or RNA. We know its exact sequence, and we’ve attached a “label” or “tag” to it so we can see it later
  • Target: The specific DNA or RNA sequence within the patient’s cells that we want to detect
  • Hybridization: After preparing the cells and denaturing the probe and target DNA to make them single-stranded, the probe is applied. It diffuses into the cells and binds only to its exact complementary sequence, like a key fitting into a specific lock

The General ISH Workflow

Performing ISH is a meticulous, multi-step process that often involves collaboration between the histology and molecular biology departments

  • Tissue/Cell Preparation: The sample (e.g., a thin slice of a tumor on a glass slide or cells from an amniotic fluid sample) is first fixed (often with formalin) to preserve the cell structure and lock the nucleic acids in place
  • Permeabilization: The cell membranes are treated (often with detergents or enzymes) to create pores, allowing the probe to enter the cell and nucleus
  • Protease Digestion: A brief treatment with an enzyme like pepsin or proteinase K helps to digest proteins that may be “masking” the target DNA, making it more accessible to the probe
  • Denaturation: The slide is heated to a high temperature (~75-95°C). This breaks the hydrogen bonds in the target DNA (in the patient’s cells) and the probe DNA, making them both single-stranded
  • Hybridization: The labeled probe is applied to the slide. The slide is then incubated at a controlled temperature (often ~37°C overnight) to allow the probe to find and anneal to its specific target sequence
  • Stringency Washes: This is a critical step! The slides are washed in special salt solutions at specific temperatures. The goal is to wash away any probe molecules that have bound non-specifically or to mismatched sequences. High stringency conditions (low salt, high temperature) ensure that only the perfectly matched probe-target duplexes remain
  • Detection & Visualization: We now need to “see” where the labeled probe has bound. This step depends entirely on the type of ISH being performed. The slide is usually counterstained with a dye like DAPI (blue) or hematoxylin (blue/purple) to make the cell nuclei visible

Major Types of ISH in the Clinical Lab

In the modern clinical lab, you’ll primarily encounter two main “flavors” of ISH, distinguished by the type of label on the probe and how it’s detected

Fluorescence In Situ Hybridization (FISH)

This is the workhorse of clinical cytogenetics and cancer diagnostics

  • The Label: The probe is directly labeled with fluorophores—molecules that emit bright, colored light when excited by a laser. Different probes can be labeled with different colors (e.g., green, red, aqua, gold)
  • Detection: The slide is viewed under a specialized fluorescence microscope equipped with filters specific for each color
  • The Result: A technologist sees colored dots, or “signals,” within the cell nuclei. By counting the signals and observing their patterns, we can make a diagnosis
  • Clinical Applications
    • Gene Amplification: Detecting extra copies of an oncogene. The classic example is testing for HER2 gene amplification in breast cancer to guide Herceptin therapy. A normal cell has two red signals (HER2 gene) and two green signals (control centromere). A cancer cell might have 10, 20, or more red signals, indicating amplification
    • Gene Rearrangements (Translocations): Using “break-apart” or “fusion” probes to detect chromosomal translocations that create cancer-causing fusion genes, like the BCR-ABL1 fusion in Chronic Myeloid Leukemia (CML)
    • Aneuploidy Detection: Rapidly counting chromosomes in prenatal samples (e.g., amniocytes) or cancer cells by using probes specific for chromosomes 13, 18, 21, X, and Y. Trisomy 21 (Down syndrome) would show three signals for the chromosome 21 probe

Chromogenic In Situ Hybridization (CISH)

CISH provides similar information to FISH but uses a different detection system that can be viewed on a standard microscope

  • The Label: The probe is labeled with a hapten like biotin or digoxigenin (DIG). This is an indirect label
  • Detection: An antibody or protein (e.g., anti-DIG or streptavidin) conjugated to an enzyme like Horseradish Peroxidase (HRP) is added. This enzyme then converts a colorless chromogenic substrate into a colored precipitate
  • The Result: Instead of glowing dots, the technologist sees small, dark colored dots (often brown or blue) under a standard bright-field microscope
  • Clinical Applications
    • CISH is also commonly used for detecting HER2 gene amplification and other gene copy number changes
    • It is excellent for detecting certain viruses, like HPV or EBV, within tissue samples
    • Advantage: The colored signal is permanent, and slides can be archived and reviewed for years. The lab doesn’t need an expensive fluorescence microscope

In conclusion, ISH is a powerful technique that bridges the gap between classic histology and molecular diagnostics. It provides crucial genetic information while preserving the cellular landscape, allowing us to see exactly where the genetic action is happening

Key Terms

  • In Situ Hybridization (ISH): A technique used to detect and localize a specific DNA or RNA sequence directly within an intact cell or tissue section, preserving the morphological context
  • Probe: A labeled, single-stranded nucleic acid molecule of a known sequence that is used to find and bind to its complementary target sequence
  • Target: The specific DNA or RNA sequence within the patient’s cells or tissue that the probe is designed to detect
  • Hybridization: The process by which two complementary, single-stranded nucleic acid molecules (e.g., probe and target) anneal to form a stable double-stranded molecule through hydrogen bonding
  • Stringency: The reaction conditions (primarily temperature and salt concentration) of post-hybridization washes that determine the specificity of probe binding. High stringency ensures only perfectly matched probes remain bound
  • FISH (Fluorescence In Situ Hybridization): A type of ISH that uses probes labeled with fluorescent dyes (fluorophores), which are visualized as colored signals using a fluorescence microscope
  • CISH (Chromogenic In Situ Hybridization): A type of ISH that uses an enzyme-based detection system to generate a colored, insoluble precipitate at the probe binding site, which is visualized with a standard bright-field microscope