Immunohistochemistry: Protocol and applications

What is Immunohistochemistry:

Immunohistochemistry (IHC) is a molecular technique used in histopathology and research to visualize the presence and distribution of specific proteins within tissue samples that usually act as biomarkers. This method involves a series of carefully design  steps to ensure accurate and reliable results. In this post, we'll go through each stage of the immunohistochemical process, from dewaxing to mounting.

 

Step 1: Dewaxing

Paraffin-embedded blocks are sectioned and placed on a glass slide to preserve tissue sections. The paraffin wax is removed from the tissue slices at the start of the procedure. The dewaxing procedure is essential for revealing the antigens in the tissue that the antibodies will subsequently target. Usually, the wax is dissolved using xylene or its replacement, which promotes the best possible antigen retrieval.

Step 2: Rehydrate

To return the tissue sections to their original form, rehydration is required after dewaxing. This introduces the sections to a range of ethanol concentrations (100%, 90%, 80%, and 70% ethanol), starting at high levels and progressively down to water. To guarantee appropriate antibody penetration and shield tissue from stress, rehydration is crucial for the following stages.

Step 3: Retrieval

To uncover and reveal antigens that might have been hidden during fixation or as a result of the effects of paraffin embedding, antigen retrieval is an essential step. A popular technique called heat-induced epitope retrieval (HIER) uses heat—typically in the form of a citrate buffer solution—to promote antigen exposure. Retrieval solutions with high or low pH might be utilized for that. For precise methodology, refer to the product description. This technique primarily uses a water bath that is heated to a high temperature of 90°C to 100°C.

Step 4: Peroxidase Block

A peroxidase block is used to reduce background staining that is not specific. To inhibit endogenous peroxidase activity, the tissue pieces are treated with hydrogen peroxide in this phase. By stopping this process, the target antigen will be specifically recognized in the ensuing immunohistochemistry reaction.

Step 5: Primary Antibody

Primary antibody attaches themselves precisely to the target antigen in the tissue. The primary antibody in this phase needs to be carefully chosen based on the particular protein of interest. Temperature and duration of incubation are critical factors in enabling the main antibody to attach to its target. Remember there are two types of antibodies one is ready to use (RTU) other is concentrated and needs to be diluted according to manufacturing guidelines.

 

Step 6: Secondary Antibody

The secondary antibody is conjugated with an enzyme, most commonly horseradish peroxidase (HRP) or alkaline phosphatase. It binds to the primary antibody and amplifies the signal, enhancing the detection of the target antigen. Excess secondary antibody is washed away to minimize background staining by wash buffer.

 

Step 7: DAB (3,3'-diaminobenzidine) Reaction

DAB is a chromogenic substrate that reacts with the enzyme conjugated to the secondary antibody, producing a visible brown precipitate at the site of the target antigen. This reaction provides a clear and contrasting signal against the background. Handle carefully because it's potentially carcinogenic.

 

Step 8: Hematoxylin Counterstain

Hematoxylin is used to counterstain the cell nuclei, providing contrast to the DAB-stained areas. This step aids in the visualization of tissue morphology and enhances the overall clarity of the immunohistochemical results.

 

Step 9: Dehydrate

After staining, the tissue sections are dehydrated by passing through a series of graded alcohols in the reverse order of rehydration (70% to 100%). Dehydration prepares the tissue for mounting.

 

Step 10: Mounting

The final step involves mounting the tissue sections onto glass slides using a mounting medium mostly toluene-free mounting media. This preserves the stained sections, allowing for long-term storage and examination under a microscope. It's important to cover very carefully to avoid air bubbles. Now Slides are observed under a microscope to observe the expression of the targeted biomarker.

Microscopy image

Applications of Immunohistochemistry

• Medical Diagnostics: Enables precise identification and characterization of proteins associated with diseases, aiding in the diagnosis and classification of various conditions, particularly in the field of oncology.
• Research Exploration: Facilitates the investigation of protein expression patterns in tissues, offering insights into cellular functions, signaling pathways, and disease mechanisms, thereby contributing to advancements in scientific knowledge.
• Pharmaceutical Development: Plays a crucial role in drug discovery and development by validating potential therapeutic targets and assessing the efficacy of novel drugs, assisting in the advancement of pharmaceutical research.
• Veterinary Medicine: Extends its utility to the realm of animal health, allowing for the study of protein expression in tissues of various species and contributing to the understanding of diseases in veterinary medicine.
• Plant Biology: Applied in the study of plant tissues to explore protein expression patterns, contributing to a deeper understanding of plant biology and providing insights into plant-specific diseases and stress responses.
• Environmental Studies: Used in environmental research to investigate protein expression in tissues of organisms exposed to different environmental conditions, aiding in the assessment of ecological impacts and potential biomonitoring applications.
• Broad Scientific Applications: Offers a versatile platform for visualizing and quantifying protein expression, making it an indispensable technique in a wide array of scientific disciplines, bridging the gap between basic research and practical applications.

Why we don’t use automatic immunohistochemistry instead of the manual? You will find the answer below table

Feature	Automatic IHC	Manual IHC
Efficiency and Throughput	Advantage: High throughput, simultaneous processing.	Advantage: Flexibility, suitable for smaller workloads.
	Limitation: Initial setup and maintenance may be complex.	Limitation: Time-consuming for larger-scale studies.
Consistency	Advantage: Greater reproducibility due to automation.	Advantage: Offers flexibility but may result in variation.
	Limitation: Requires standardized protocols.	Limitation: Variability between operators.
Precision	Advantage: Precise control over staining parameters.	Advantage: Allows adaptability but may lack precision.
	Limitation: Initial optimization required.	Limitation: Greater susceptibility to human error.
Labor Requirements	Advantage: Requires less hands-on time.	Advantage: Suited for varying skill levels.
	Limitation: Skilled personnel are needed for setup.	Limitation: Time-consuming for optimization.
Flexibility	Advantage: Limited adaptability to unconventional methods.	Advantage: Adaptable to specific experimental needs.
	Limitation: May not suit unconventional staining.	Limitation: Standardization can be challenging.
Cost	Advantage: Higher initial investment, lower labor costs.	Advantage: Generally more cost-effective upfront.
	Limitation: Consumables and maintenance costs.	Limitation: Consumables costs may still accumulate.
Skill Level	Advantage: Skilled personnel needed for setup.	Advantage: Suited for varying skill levels.
	Limitation: Less operator skill required.	Limitation: Consistency may depend on operator skill.
Adaptability	Advantage: May not adapt well to unconventional methods.	Advantage: Easily adaptable to specific requirements.
	Limitation: Limited flexibility in certain situations.	Limitation: Requires adherence to standardized protocols.

The immunohistochemical process from dewaxing to mounting is essential for obtaining accurate and reliable results in pathology and research. Each step plays a crucial role in ensuring the specificity and sensitivity of the immunohistochemical reaction, ultimately allowing researchers and pathologists to gain valuable insights into the distribution of proteins within tissues. Immunohistochemistry is no doubt a cheaper and more effective molecular technique that provides efficient results.