Last Updated on October 14, 2025 by Analgesia team
IHC full form
IHC stands for immunohistochemistry, a laboratory technique for identifying and visualising antigens in tissue samples. IHC is a laboratory technique that combines histology and immunology. It involves
- Placing a tissue sample on a glass slide
- Adding an antibody that binds to a specific target
- Staining the sample with an enzyme or dye
- Viewing the sample under a microscope
Purpose Of IHC
The purpose of immunohistochemistry (IHC) is to help diagnose diseases by detecting antigens in tissue samples:
- Cancer: IHC is used to diagnose cancer, predict treatment response, and determine prognosis. It can help identify the type of cancer, where it started, and how likely it is to grow.
- Neurological conditions and blood disorders: IHC can help classify these conditions and blood disorders.
- Brain trauma: IHC can detect axonal injury within hours of a head injury. This can help establish the timing of the injury in medico-legal settings.
Procedure
The immunohistochemistry (IHC) procedure involves several steps, including:
- Antigen retrieval
This step is often required for formalin-fixed tissues. It reverses the masking of epitopes and restores the binding between epitopes and antibodies. If omitted, this step can result in weak or false-negative staining.
- Primary antibody incubation
The primary antibody is incubated on the tissue sample, usually for one hour, but sometimes overnight. The antibody binds to the antigen if it’s present.
- Washing
Excess unbound primary antibody is washed away.
- Secondary antibody incubation
The tissue is incubated with a labelled secondary antibody. This typically takes 30 minutes to one hour at room temperature or 37°C.
- Counterstaining
To visualise specific structures in the tissue or cells, they are stained in contrasting colors. Hematoxylin or methyl green are commonly used for nuclear staining.
- Mounting
The sample is mounted to prevent drying and fading, and to make it suitable for long-term storage. Water-soluble or hydrophobic mounting media can be used
Immunohistochemistry (IHC) has many advantages, including:
Visualises antigens:
IHC allows scientists and pathologists to detect specific antigens within cells and tissues using targeted antibodies. This makes it possible not only to confirm the presence of certain molecules but also to pinpoint exactly where they’re located within the tissue sample. Such precision is essential for identifying abnormal protein expression in diseases like cancer.
Preserves tissue structure:
Unlike some biochemical techniques that destroy cell architecture, IHC keeps the overall tissue structure intact. This lets pathologists observe how a particular molecule is distributed in relation to other cells and structures, helping them understand the disease within its natural microenvironment.
Permanent record:
The staining produced by IHC is stable and long-lasting, meaning the results can be stored and reviewed years later. This permanent record is invaluable for follow-up studies, audits, or second opinions in clinical diagnostics.
Widely used:
IHC is one of the most commonly used tools in modern pathology. It plays a vital role in diagnosing cancers, infections, and autoimmune diseases by revealing the molecular characteristics of tissues. Its reliability and diagnostic accuracy make it a standard method across hospitals and research laboratories worldwide.
Versatile:
The technique can be applied to a wide range of tissue types, including fresh and frozen specimens. It also works seamlessly with routine histological samples preserved in formalin and embedded in paraffin. This flexibility makes IHC suitable for both clinical and research applications.
Low cost:
Compared to molecular diagnostic techniques like PCR or next-generation sequencing, IHC is relatively inexpensive. The reagents, antibodies, and equipment required are widely available, making it a cost-effective choice for many laboratories, especially in resource-limited settings.
Quick turnaround:
IHC delivers results quickly, often within a few hours to a day. This speed is crucial for patient management, particularly when rapid diagnosis can influence treatment decisions, such as in cancer care or infectious disease outbreaks.
Minimal risk:
The process poses minimal risk to laboratory personnel because it does not involve handling live or infectious materials. Tissue samples are typically fixed and inactivated before staining, ensuring a high level of biosafety.
Detects infectious organisms:
IHC can identify specific infectious organisms directly within tissue samples. By using antibodies that bind to pathogen-specific antigens, it can confirm infections caused by bacteria, viruses, fungi, or parasites—especially when traditional cultures fail.
Detects cellular changes:
The technique can reveal subtle cellular alterations and molecular changes triggered by disease or exposure to chemicals. This makes it a valuable tool for toxicology studies, cancer research, and drug development, where understanding cellular response is key.
Detects axonal injury:
IHC can identify signs of axonal damage within hours of a head injury. This sensitivity helps forensic and clinical pathologists detect early nerve damage that might not yet be visible through conventional microscopic examination.
Limitations of immunohistochemistry (IHC)
Subjectivity in interpretation:
The accuracy of IHC results often depends on the pathologist’s experience and interpretation. Staining intensity, background colour, and tissue artefacts can all influence how results are read. Without proper controls, what appears as positive staining could sometimes be misleading.
Antibody specificity and cross-reactivity:
Not all antibodies are perfectly specific. Some may bind to unintended targets, leading to false-positive or false-negative results. Careful antibody validation and optimisation are essential, but these steps can be time-consuming and technically demanding.
Limited quantification:
While IHC provides excellent visualisation, it is not fully quantitative. The staining intensity offers only an approximate measure of protein expression. More precise quantification usually requires additional techniques such as Western blotting or ELISA.
Technical variability:
Differences in tissue fixation, antigen retrieval methods, and staining protocols can cause inconsistent results between laboratories. Even small variations in incubation time or temperature can alter the final staining pattern, making standardisation a challenge.
Antigen masking:
Over-fixation of tissue in formalin can mask antigen sites, making them inaccessible to antibodies. Although antigen retrieval techniques can reverse some of this masking, not all antigens recover fully, reducing sensitivity.
Limited sensitivity for low-abundance proteins:
IHC may struggle to detect proteins that are present in very small quantities. In such cases, more sensitive molecular techniques might be required to confirm the findings.
Time-consuming optimisation:
Each new antibody or tissue type often requires trial and error to determine the ideal conditions for staining. This optimisation process can delay diagnostic or research timelines, particularly in complex studies.
Potential for background staining:
Antibodies’ nonspecific binding can lead to unwanted background staining, which complicates interpretation. This is especially problematic when the target antigen is weakly expressed or when tissues contain high levels of endogenous enzymes.
Dependency on good tissue preservation:
The quality of IHC results heavily depends on how well the tissue sample has been preserved. Poor fixation or delayed processing can cause proteins to degrade, leading to patchy or unreliable staining patterns.
Limited multiplexing capability:
Although advances are being made, traditional IHC usually allows detection of only one or a few markers at a time. This can be a drawback when studying complex tissues that require simultaneous analysis of multiple biomarkers.
Cost and availability of high-quality antibodies:
While IHC itself is cost-effective, sourcing high-quality, specific antibodies for rare targets can be expensive. Poor-quality reagents increase the risk of inaccurate results and wasted resources.