Immunohistochemistry (IHC) is a technique used in laboratory diagnostics to identify specific proteins within tissue cells. This method provides targeted information that complements traditional staining methods, which primarily show tissue structure. The insights gained from IHC are applied in both medical diagnostics and various areas of scientific research.
The Principle of Immunohistochemistry
Immunohistochemistry operates on the specific binding that occurs between an antibody and its corresponding antigen, a target protein within a cell. This interaction is often compared to a lock and key, where a specific antibody is designed to recognize and attach only to its specific antigen. This specificity allows scientists and clinicians to pinpoint the exact location of a protein within a complex tissue sample.
The IHC procedure begins with preparing the tissue. Samples are preserved through fixation, sliced into extremely thin sections, and mounted on microscope slides. A primary antibody is then introduced to the tissue, where it binds to the target antigen. Following this, a secondary antibody, which is engineered to bind to the primary antibody, is applied to amplify the signal and increase the sensitivity of the test.
Attached to the secondary antibody is an enzyme, such as Horseradish Peroxidase (HRP). In the final step, a substance called a chromogen is added, which the enzyme reacts with. This reaction causes the chromogen to change color and deposit at the site of the antigen-antibody complex. This colored precipitate creates a visible stain under a microscope, mapping the protein’s distribution, while a counterstain provides context by coloring the rest of the tissue.
Leica’s Automated IHC Solutions
The transition from manual to automated IHC addresses challenges related to consistency, labor, and turnaround time. Leica Biosystems’ BOND series of automated systems, such as the BOND-III and BOND-MAX, standardize the IHC workflow. These “walk-away” instruments mean a technician can load slides and reagents, start a run, and return when the staining is complete. This automation reduces human error and ensures each slide is processed with a consistent protocol.
Inside a BOND instrument, robotic components precisely control every step of the IHC process. The system begins with on-board slide baking and deparaffinization, which prepares the tissue by removing paraffin wax. It then performs antigen retrieval, a process using heat and specialized solutions to unmask target proteins and make them accessible to antibodies. Automating this step is a major advantage as it can significantly impact results.
The machine’s robotic arms dispense precise volumes of reagents, including primary antibodies and detection systems. Incubation times and temperatures are tightly regulated for optimal binding conditions. Between each step, the system performs automated washing cycles to remove unbound reagents, which prevents background staining and produces a clean result. This control allows laboratories to achieve highly reproducible outcomes.
Essential Reagents and Detection Systems
The quality of an IHC stain depends on the chemical reagents used. Leica provides a portfolio of reagents designed to function as an integrated system with its automated instruments. A component of this is the line of Novocastra primary antibodies, which are developed and validated for use on the BOND platforms. This helps ensure they perform reliably and provide consistent results.
To complement the primary antibodies, Leica offers detection kits like the BOND Polymer Refine Detection system. This ready-to-use kit contains the secondary antibody, an amplifying polymer with its attached enzyme, and the chromogen. Using a pre-optimized detection system ensures all chemical components are compatible. This polymer-based system amplifies the signal, leading to more intense staining by increasing the number of enzyme molecules at the target site.
The system is supported by a range of ancillary reagents. These include specific antigen retrieval solutions needed to prepare different tissue types, as well as buffers for washing and mounting media for preserving the finished slide. By providing a complete ecosystem of instruments and reagents, the company creates a streamlined workflow that helps laboratories meet quality assurance standards.
Clinical and Research Applications
The application of IHC technology has a significant impact in both clinical diagnostics and scientific research. In oncology, it is a foundational tool for diagnosing and characterizing cancer. Pathologists use IHC to differentiate between various types of tumors that may appear similar but have different protein expression profiles. This information is important for determining the aggressiveness of a cancer and guiding treatment.
A prominent example is in the diagnosis of breast cancer. IHC tests are used to determine the status of estrogen receptors (ER), progesterone receptors (PR), and the human epidermal growth factor receptor 2 (HER2). The presence or absence of these protein biomarkers directly informs oncologists about which therapies, such as hormone therapy or targeted drugs, are most likely to be effective for a patient.
Beyond cancer, IHC is used to identify infectious agents, such as viruses, directly within tissue sections. In neuroscience, researchers use IHC to map the distribution of specific proteins in the brain. This helps in the study of neurodegenerative diseases like Alzheimer’s and Parkinson’s by revealing how protein abnormalities contribute to disease progression. Pharmaceutical companies also utilize IHC in drug development to verify that a new therapeutic is interacting with its intended protein target.