Immunohistochemistry (IHC) is a laboratory method that allows scientists and medical professionals to visualize specific biological components, such as proteins or antigens, directly within a tissue sample. The technique uses antibodies to identify and pinpoint the exact location of these target molecules within cells and tissues. IHC combines principles of immunology (the study of antibodies) with histology (the study of tissue structure) to reveal the precise presence and distribution of a chosen protein in its natural context, offering molecular specificity unlike general tissue stains.
The Core Mechanism of Staining
The fundamental principle behind IHC is the highly specific lock-and-key interaction between an antibody and its target molecule, known as an antigen. In IHC, the antigen is the specific protein of interest, often one associated with a disease. The laboratory-manufactured antibody is designed to recognize and bind only to a unique site, or epitope, on that target antigen.
Once the primary antibody binds to the antigen, a detection system is needed to make this invisible binding event visible under a microscope. This is often achieved using a secondary antibody tagged with a reporter molecule, such as an enzyme. The secondary antibody specifically recognizes and attaches to the primary antibody, creating strong signal amplification.
A common reporter enzyme is Horseradish Peroxidase (HRP), which reacts with a colorless chemical substrate called a chromogen, such as DAB. This reaction produces an insoluble, intensely colored product—often a brown precipitate—at the exact site of the antigen’s location. This localized color change allows pathologists to see where the protein is expressed within the tissue.
Step-by-Step Procedure
The IHC process begins with meticulous tissue preparation to preserve the sample’s structure and the target protein’s integrity. The tissue, often obtained through a biopsy, is typically preserved by chemical fixation, most commonly using formalin. This fixation cross-links proteins to prevent degradation. The fixed tissue is then embedded in a solid medium, usually paraffin wax, which provides structural support for slicing the sample into extremely thin sections.
These thin sections are placed onto glass slides and must undergo antigen retrieval, which “unmasks” the target protein. Fixation can hide the antigen’s binding site, so heat-induced epitope retrieval (HIER) uses heat to break these bonds, making the antigen accessible. Following retrieval, a blocking step is performed using an innocuous protein solution to occupy any non-specific binding sites on the tissue.
The slides are then incubated with the primary antibody, allowing it to bind specifically to the target antigen present in the tissue. After washing away unbound primary antibody, the tagged secondary antibody is applied, which attaches to the primary antibody. Finally, the chromogen substrate is added to react with the enzyme tag, producing the colored stain. A counterstain, like Hematoxylin, is used to lightly stain the cell nuclei blue, providing structural context for the specific stain.
Primary Applications in Medicine
Immunohistochemistry is an indispensable tool in clinical pathology, primarily used for the diagnosis and classification of cancer. Pathologists use IHC to differentiate between benign and malignant tumors and to determine the specific type of cancer present. This is important for classifying tumors that look similar under a standard microscope or for identifying the tissue of origin for a metastatic cancer.
A highly practical example is in breast cancer, where IHC tests for the expression of hormone receptors like Estrogen Receptor (ER) and Progesterone Receptor (PR), as well as the HER2 protein. The results of these tests determine if a tumor is hormone-sensitive or if it is a candidate for targeted therapies. This information profoundly influences the patient’s treatment plan. Similarly, in melanoma, IHC can detect markers like Melan-A or HMB-45 to confirm the diagnosis.
Beyond oncology, IHC is used to identify infectious agents in tissue samples by using antibodies that target specific viral or bacterial proteins. For instance, it can detect the presence of Cytomegalovirus or Mycobacterium tuberculosis in infected tissues. The technique is also applied in neuroscience to visualize abnormal protein accumulations, such as the amyloid-beta plaques and tau tangles that are hallmarks of Alzheimer’s disease.
Interpreting the Stained Tissue
A pathologist interprets the final stained slide by examining the tissue under a light microscope, looking for the presence and characteristics of the colored precipitate. Interpretation involves assessing three main visual criteria: location, intensity, and percentage of positive cells. The location of the stain is significant, as a protein’s function is often tied to where it resides, such as in the cell nucleus, cytoplasm, or cell membrane.
The intensity of the color, graded from weak to strong, indicates the level of protein expression within the cell. A strong, dark color suggests high expression, while a faint color indicates low expression. Pathologists also determine the percentage of cells that exhibit positive staining within the tumor area. Many diagnostic scoring systems combine these factors—intensity and percentage—to generate a score that translates into a clinical conclusion.