Immunohistochemistry (IHC) is a technique used for detecting antigens in biological specimens using the principle of antigen-antibody interactions. Tumour tissues are embedded on the glass slide, and light microscopy is used for analysis. IHC is used extensively in research and clinical diagnostics to visualize the proper distribution of cellular components within the cell. It has been instrumental in classifying neoplasms and determining the origin of metastatic tumours (Megaki et al.; 2019). In this article, we go over IHC in greater depth.


The procedure starts with the sample collection and processing, which is crucial as the antigen exhibition and location largely depend on the quality of tissue samples. The steps are briefly described as follows:

1. Antigen Retrieval: involves the pre-treatment of tissue to retrieve antigens masked by fixation and make them more accessible to antibody binding.
2. Primary antibody: Antibodies (monoclonal or polyclonal) are added, which bind to the specific antigen under investigation. Monoclonal antibodies bind to a single antigenic epitope, and polyclonal antibodies bind to multiple antigenic domains (Megaki et al.; 2019).

3. Secondary antibody: A labelled second antibody targeted against the primary antibody is added to visualize antigen-antibody interaction using light microscopy.

4. Detection The detection buffer allows localizing of the primary antibody.


IHC is an essential tool in cancer diagnostics. It plays a vital role in pathology, especially in oncologic pathology, neuropathology, and hematopathology (Duraiyan et al.; 2012).

Following are some of the applications of IHC:

1. Cancer Diagnostics: Clinicians utilize this technology to discover whether a tumour is benign or malignant using tumour-specific markers. It helps determine the stage and grade of cancer origin of metastasis to find the primary tumour site (Duraiyan et al.; 2012).

2. Identification of Tumours of Uncertain Histogenesis: IHC is highly advantageous in diagnosing a tumour of unknown origin. A panel of antibodies is selected basis the clinical history, morphological features, and other relevant test reports used to resolve such diagnostic problem cases (Duraiyan et al.; 2012).

3. Response to therapy: IHC can also predict the therapeutic response of breast and prostate cancers. The growth of these tumours is regulated by the hormones oestrogen and androgen, respectively. The receptors for these growth hormones are found on the tumour cells themselves. Tumours expressing a high level of receptor positivity would benefit from removing the endogenous source of such hormones or from hormonal therapy to lower their levels – oestrogen therapy in prostate cancer and androgen therapy in breast cancer (Duraiyan et al.; 2012).

4. Infections: Another critical application of Immunohistochemical methods is to identify the infectious agent in tissues using specific antibodies against microbial DNA or RNA. It can also help identify microorganisms in cytological preparations like fluids, sputum and needle aspiration specimens (Duraiyan et al.; 2012).

5. Genetics: The role of a particular gene product in a biological process can also be determined using IHC like development and apoptosis (Duraiyan et al.; 2012).


Jeyapradha Duraiyan, Rajeshwar Govindarajan,Karunakaran Kaliyappan, and Murugesan Palanisamy. Applications of immunohistochemistry. J Pharm Bioallied Sci. 2012; 4(Suppl 2): S307–S309
Polymerase Chain Reaction (PCR) [WWW Document], n.d.. Genome.gov. URL https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction (accessed 2.18.22).

Shino Magaki, Seyed A. Hojat, Bowen Wei, Alexandra So, William H. Yong. An Introduction to the Performance of Immunohistochemistry. Methods Mol Biol. 2019; 1897: 289–298