Immunohistochemistry (IHC) is a technique that unveils the precise localization of proteins within tissue sections, providing a marriage of morphological and molecular insights.  It involves the use of antibodies to bind to target proteins in situ, allowing for their detection and visualization under a microscope. This technique has become indispensable in diagnostic pathology and biomedical research. This article journeys through the meticulous process of IHC, from sample preparation to staining, illuminating the technical finesse required to reveal the cellular and molecular architecture of tissues.

Here’s a step-by-step guide from sample preparation to staining in IHC:


Figure Source: Wikipedia-Main staining patterns on chromogenic immunohistochemistry.

Step1: Sample Preparation

Tissue Collection and Fixation: The first step in IHC is tissue collection, followed by fixation to preserve tissue morphology and molecular integrity. Common fixatives include formalin and paraformaldehyde. Fixation times vary but typically range from hours to overnight.

Embedding and Sectioning: After fixation, the tissue is dehydrated through a series of alcohol solutions and then embedded in paraffin wax or frozen in optimal cutting temperature (OCT) compound. Paraffin embedding is common for formalin-fixed tissues, while OCT embedding is used for frozen sections. Thin sections (usually 4-5 μm thick) are then obtained using a microtome or cryostat for paraffin or frozen sections respectively.


Step 2: Antigen Retrieval Heat-induced Epitope Retrieval (HIER)

To unmask antigens masked during fixation, heat-induced epitope retrieval using citrate buffer or EDTA is employed, enhancing antigen-antibody binding.

Step 3 : Blocking and Primary Antibody Incubation

*Blocking: Blocking non-specific binding sites using normal serum or proteins like bovine serum albumin (BSA) is crucial to reduce background staining.

*Primary Antibody Incubation: The tissue sections are incubated with primary antibodies specific to the antigen of interest. The choice of antibody and its concentration are pivotal for specific and sensitive detection. The incubation time and temperature can vary but typically range from 30 minutes to overnight.

Step 4 : Detection and Visualization

*Secondary Antibody and Chromogen Incubation: After washing to remove unbound primary antibodies, secondary antibodies conjugated to enzymes like horseradish peroxidase (HRP) or alkaline phosphatase (AP) are applied, followed by chromogen substrates to visualize the antigen-antibody complex. Depending on the detection method, the signal can be visualized by various techniques, such as fluorescence microscopy, chromogenic detection (using enzyme-substrate reactions), or silver enhancement.

*Fluorescence IHC: Alternatively, fluorescence IHC employs fluorophore-conjugated secondary antibodies to visualize the antigens under a fluorescence microscope.

Counterstaining, Mounting, and Imaging

*Counterstaining: Hematoxylin is often used as a counterstain to provide a morphological context by staining the cell nuclei.

*Mounting and Imaging: Finally, the sections are mounted with a suitable medium, and images are captured using a light or fluorescence microscope to visualize the localization and distribution of the target protein.

The success of an IHC experiment depends on careful optimization of each step, including antibody selection, fixation, antigen retrieval, and detection methods. Proper controls, including negative controls (omitting primary antibodies) and positive controls (known positive tissues), are essential to ensure the specificity and reliability of the staining results.

Immunohistochemistry (IHC) stands as a testament to the blend of technological innovation and biological insight, enabling the visualization and localization of specific proteins within tissue sections. Its applications span across diagnostics, research, and therapeutic evaluation, making it a cornerstone in modern pathology and biomedical research. The market surrounding IHC has been burgeoning, driven by the demand for precise diagnostics and therapeutic monitoring.

The IHC market is currently on an upward trajectory, buoyed by technological advancements, growing prevalence of chronic diseases, and rising investments in R&D activities. Prominent players like Abcam plc, Agilent Technologies, Inc., Bio-Rad Laboratories, Inc., and PerkinElmer, Inc., are continually innovating and expanding their IHC product portfolios. Geographically, North America and Europe are leading in the IHC market due to substantial investments in healthcare and research. However, the Asia-Pacific region is anticipated to exhibit rapid growth driven by rising healthcare expenditure and growing awareness about advanced diagnostic technologies.

The market is poised for steady growth with a CAGR (Compound Annual Growth Rate) projected to remain positive over the next decade. The expansion of personalized medicine, coupled with advancements in automated IHC platforms, is expected to propel the market further. The myriad applications of Immunohistochemistry underscore its indelible impact on modern medicine and research. As the market continues to expand, driven by technological innovations and growing healthcare needs, IHC is set to remain a pivotal tool, delivering invaluable insights at the cellular and molecular level. The evolving market dynamics augur well for stakeholders, promising a sustained growth and broader adoption of IHC across the global healthcare and research landscape.

KMD Bioscience offers comprehensive IHC services from project design, marker selection to image completion and data analysis. KMD Bioscience’s experienced scientists have over 10 years of experience in IHC and IF services, and KMD Bioscience’s scientists will work closely with customers to provide high-quality services. KMD Bioscience started from the phage display technology platform to provide high-quality antibody discovery services, and gradually established stable cell line construction and screening, natural protein extraction and fermentation, and recombinant protein custom expression services, a multi-species antibody discovery platform.

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