Immunohistochemistry (IHC) is a laboratory technique used to identify specific molecules, primarily proteins, within tissue samples. Its purpose is to visualize the presence and location of these proteins, creating a “cellular map” that helps in understanding cellular processes and diagnosing diseases. The principle of IHC can be compared to using a specific key (an antibody) to find and fit into a unique lock (an antigen) within a tissue sample. This method allows pathologists and researchers to see what is happening inside cells, providing detailed information that is not visible with standard microscopy.
The Core Mechanism of IHC
The foundation of IHC is the highly specific interaction between an antigen and an antibody. An antigen is a protein within a cell that serves as a target, while an antibody is a protein produced to recognize and bind exclusively to that target. This binding is precise, ensuring the antibody attaches only to the protein of interest among thousands of others within the tissue.
To make this interaction visible, the antibody is linked to a label, often an enzyme like horseradish peroxidase (HRP) or a fluorescent molecule. After the antibody has bound to its target antigen in the tissue, a substrate is introduced. If an enzyme label is used, the substrate triggers a chemical reaction that produces a colored precipitate at the exact location of the antibody-antigen complex.
This color, commonly brown or red, can be seen with a standard light microscope. This process “stains” only the cells containing the target protein, revealing their location, distribution, and abundance.
The IHC Laboratory Process
The process from a tissue sample to a stained slide involves several steps to preserve the tissue and make target proteins accessible. First, tissue from a biopsy is preserved through a process called fixation, using a chemical like formalin. Fixation maintains the tissue’s structure, and the fixed tissue is then embedded in paraffin wax, forming a solid block that can be handled easily.
The wax block is cut into extremely thin sections using an instrument called a microtome and placed onto glass microscope slides. Before staining can begin, the wax is removed from the tissue in a step called deparaffinization, followed by rehydration.
Antigen retrieval is then performed. The fixation process can mask the target antigens, so this step uses heat or enzymes to unmask these sites, allowing antibodies to bind. The slide is then incubated with the primary antibody, which specifically attaches to the target protein. Following this, a secondary antibody, which is engineered to bind to the primary antibody and carries the enzyme label, is applied.
Finally, a substrate-chromogen solution is added, which the enzyme on the secondary antibody converts into a colored deposit, making the target visible. To provide context, a counterstain, such as hematoxylin, is applied. It stains the nuclei of all cells blue, creating a contrast that makes the specific stain from the IHC reaction stand out.
Clinical and Research Applications
IHC is used widely in both clinical diagnostics and scientific research. In medicine, its most frequent application is in oncology for cancer diagnosis and management. Pathologists use IHC to differentiate between types of cancer that may look similar and to determine the origin of metastatic cancer, where cancer has spread from its primary site.
For example, in breast cancer diagnostics, IHC tests for estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2). The results of these tests help oncologists select the most effective treatment, such as hormone therapy for ER-positive cancers. Beyond cancer, IHC helps identify infectious agents, like viruses or bacteria, and aids in the diagnosis of conditions such as Alzheimer’s disease.
In research, IHC is used for studying biological processes. Scientists use it to map the location of proteins during embryonic development, investigate how diseases alter cellular components, and trace neural pathways in the brain. It allows researchers to test the effectiveness of new drugs by observing how they affect the levels or activity of specific proteins in target tissues.
Understanding IHC Results
Interpreting an IHC-stained slide involves analysis by a pathologist, who examines several features to provide a report. The first determination is whether the result is positive or negative. A positive result, indicated by the colored stain, confirms the target protein was detected, while a negative result means the protein was not found.
The location of the stain within the cell is also noted, as this provides functional clues. A protein’s location—whether in the nucleus, the cytoplasm, or on the cell membrane—is linked to its specific role and can be characteristic of certain diseases. The pattern of staining can reveal whether a protein is functioning correctly or is misplaced within the cell.
The results are quantified using a semi-quantitative scoring system. This system assesses both the intensity of the stain (graded on a scale from 0 for no stain to 3+ for strong) and the percentage of cells that show a positive stain. This combined score provides a detailed picture of the protein’s expression level, which can be linked to prognosis and guide clinical decisions.