Immunohistochemistry (IHC) Protocol and FAQs

Immunohistochemistry (IHC) is a technique widely used in cell biology and pathology. It realizes the localization and quantitative analysis of specific proteins, antigens or molecular markers by using the specific binding between antibodies and target antigens and combining morphological analysis in tissue sections. IHC can not only be used to study the mechanism of disease, but also play an important role in cancer diagnosis, marker molecular localization, protein function research and so on.

The Principle of Immunohistochemistry

Tissue sections are usually fixed with chemical reagents such as formalin during the fixation process, which may cause the antigen epitope to be obscured. Therefore, in IHC experiments, it is first necessary to expose the epitopes of the antigen through antigen repair (such as heat-induced antigen repair or enzyme-induced repair). Through this process, the cross-linked molecules in the immobilization process can be removed, the three-dimensional structure of the antigen can be restored, and the antibody binding can be facilitated. The core of the immunohistochemical experiment is the antigen-antibody reaction. By applying a specific primary antibody (primary antibody), it can bind to the target antigen in the section to form an antigen-antibody complex. In order to enhance the signal and improve the sensitivity of the detection, secondary antibodies (secondary antibodies) were used in IHC experiments. The secondary antibody is an antibody against a primary antibody-derived species (such as rabbit anti-mouse antibodies), and a marker (such as an enzyme or a fluorescent dye) is usually attached to the secondary antibody. Through the combination of secondary antibody and primary antibody, markers can be attached to the antigen-antibody complex to amplify the signal and facilitate subsequent detection. Commonly used markers include horseradish peroxidase (HRP) and alkaline phosphatase (AP). The markers on the secondary antibody generate visible signals through specific reactions. If the marker is an enzyme, after the secondary antibody binds, the enzyme reacts with the corresponding substrate to form a precipitate or a color reaction, thereby observing the position of the marker under a microscope. The common enzyme substrate reaction is the DAB (3,3 ' -diaminobenzidine) reaction, which can generate brown precipitate and clearly show the location of the antigen. In some cases, the marker can be a fluorescent dye, which is then observed using a fluorescence microscope to generate a fluorescence signal at a specific wavelength. In IHC experiments, nuclear dyes such as hematoxylin are usually used as contrast staining to distinguish the nucleus from the cytoplasm, so as to more clearly locate the location of the antigen.

The Steps of Immunohistochemistry

Sample Preparation

The tissue samples were taken out, fixed properly (such as formalin fixation), and then embedded in paraffin. To make tissue sections, the common thickness is 4-6 microns, and the sections need to be placed on the slide. Deparaffinization is performed using xylene or other deparaffin solvents to remove paraffin wax, and gradually washed with ethanol, and finally washed with distilled water.

Antigen Repair

The fixation process may cover the epitopes of some antigens, so antigen repair is required. Commonly used methods include heat-induced antigen repair (HIER) and enzyme-induced repair. The HIER method is to place the slice in a high-temperature buffer (such as citric acid buffer), and activate the antigen epitope by high temperature. Enzyme-induced repair (EIER) uses specific enzymes to remove antigen masking caused by cross-linking or fixation processes in tissues, thereby restoring the epitope of the antigen and enabling it to be recognized and bound by specific antibodies. This method is especially suitable for those antigens that cannot be restored by traditional heat-induced antigen repair (HIER), especially some cell surface markers or more fragile antigens.

Blocking Non-specific Binding

The sections were treated with blocking solution (such as a solution containing bovine serum albumin (BSA) or normal goat serum) to avoid non-specific binding of secondary antibodies.

Incubation of primary antibody

Incubating the specific primary antibody with the slice, the primary antibody will specifically bind to the target antigen in the slice. The incubation time and temperature need to be optimized according to the characteristics of the primary antibody.

Secondary Antibody Incubation

After incubation with the primary antibody, the secondary antibody corresponding to the primary antibody species is used for incubation, and the secondary antibody is usually combined with the marker (such as enzyme or fluorescent dye) for subsequent signal amplification.

Color Reaction

Common color development methods include HRP (horseradish peroxidase) binding to the substrate to form a color product, or using a fluorescently labeled secondary antibody for fluorescence microscopy. The coloring process will enlarge the staining of the antigen position, so that it can be seen.

Nuclear Contrast Staining

Hematoxylin is usually used for nuclear staining to compare the location of antigens and nuclei under a microscope.

Sealing

After staining, the sections were sealed with sealant for microscopic observation. The choice of sealing glue should ensure the long-term preservation of dyeing results.

Microscopic Observation and Analysis

The processed tissue sections were observed by microscope to analyze the location, expression and distribution of antigens. According to the staining intensity and tissue morphology, combined with quantitative analysis (such as image analysis software) for further evaluation.

Figure 1. Diagramshowingmechanismof each of the mIHC/IF platform. (A) DISCOVERY ULTRA system: after primary antibody incubation, a secondary antibody labelled with HRP is introduced. The HRP is reacted with an appropriate substrate bound to a chromogenic dye, leading to the precipitation of insoluble, coloured precipitates at the site where the antigens are found. (B) Metal-based IHC techniques such as IMC and MIBI: a primary antibody bound to the target antigen is tagged with a metal isotope of known molecular mass. Analysis is carried out using mass spectrometry in MIBI and laser ablation coupled to mass cytometry in IMC. (C) Vectra: after primary antibody incubation, a secondary antibody labelled with HRP is introduced. A fluorophore-conjugated tyramide molecule serves as the substrate for HRP, resulting in an antigen-associated fluorescence signal. (D) Nanostring’s DSP: the target antigen will bind the primary antibody which is coupled to a photocleavable oligonucleotide tag. UV light is used to cleave the oligonucleotide tags and is collected using a microcapillary tube and stored in a microplate well. The oligonucleotide tags will bind to the reporter probe via the target-specific capture probe. Reporter probes are imaged and counted by the nCounter analysis system. Abbreviations: mIHC/IF, multiplex immunohistochemistry/immunofluorescence; HRP, horseradish peroxidase; IHC, immunohistochemistry; IMC, Imaging Mass Cytometry; MIBI, Multiplexed Ion Beam Imaging; DSP, Digital Spatial Profiling. (Reference source: Overview of multiplex immunohistochemistry/ immunofluorescence techniques in the era of cancer immunotherapy.）

Advantages of Immunohistochemistry

High specificity : Through the specificity of the antigen-antibody reaction, the target molecule can be accurately positioned at the tissue level.

Morphological information : IHC not only provides information at the molecular level, but also combines the morphological characteristics of histological sections to provide tissue or cell-level localization analysis.

Multiple detection : multiple staining can be performed by using different antibodies and combining different markers to detect the expression and localization of multiple antigens.

Application of Immunohistochemistry

Cancer Diagnosis

IHC can help identify specific molecular markers in cancer cells for cancer classification, staging and prognosis evaluation.

Pathological Research

IHC is used in the field of pathology to analyze different types of tissues and cells to assess the characteristics and mechanisms of certain diseases.

Immunological Research

IHC can be used to study the function of the immune system and the interaction between cells. It can help to identify specific immune cell subsets, evaluate immune response and its relationship with disease.

Study on Disease Markers

By labeling specific antigens, IHC can help discover new disease markers. For example, it can be used to study inflammatory responses or molecular markers of certain bacterial and viral infections.

Drug Development and Clinical Trials

IHC is used to evaluate the effects of new drugs on cells or tissues during drug development, especially by observing changes in the expression of specific proteins. In addition, it is also used in clinical trials to monitor patients ' response to drugs.

Alpha Lifetech ensures high-quality, reproducible results by meticulously optimizing each assay for the specific antibodies and tissue types used. They cater to a wide range of research needs, from basic science to translational and clinical studies. Alpha Lifetech's commitment to precision and excellence makes them a trusted partner for researchers seeking to advance their understanding of biological systems and improve healthcare outcomes.

FAQ

1. Non-specific staining often occurs in IHC experiments, that is, antibodies may bind to other molecules other than the target, resulting in too strong background staining and affecting the interpretation of the results.

Solutions : (1) Appropriate control groups were used : including negative controls (no primary or secondary antibody was added) and positive controls (tissues known to express the target protein) to confirm the specificity of staining. (2) Optimizing antibody concentration : Reducing the concentration of the antibody can sometimes reduce non-specific binding. It is recommended to use the minimum effective concentration. (3) Blocking background : Using appropriate blocking agents (such as normal serum, BSA, etc.), adjusting the concentration of the blocking agent, blocking the non-specific binding sites on the section. (4) Improve washing steps : Increase the number of washings or extend the time to remove unspecifically bound antibodies.
2. The target protein may not be detected in IHC experiments It may be due to the staining signal is too weak or no signal at all.

Solutions : (1) Check the effectiveness of the antibody : ensure that the binding of the antibody to the target protein is effective and meets the experimental requirements. Try antibodies from different sources. (2) Prolonging antibody incubation time : Appropriately extending the incubation time of the antibody can enhance the signal. (3) Optimize antibody concentration : Sometimes reducing antibody concentration (especially primary antibody) helps to reduce non-specific binding, thereby increasing the intensity of the signal. (4) Optimize the antigen repair step : If the target protein is hidden in the tissue section due to the fixation process, using an appropriate antigen repair method can help restore its detectability.
3. The poor quality of tissue sections (such as too thin, too thick, tissue damage, etc.) may affect the staining effect.

Solutions : (1) Slice thickness optimization : 4-6μm tissue sections are usually recommended for IHC experiments. Too thin sections may lose the target protein, while too thick sections may lead to uneven staining. (2) Correct fixation of tissue : use the appropriate fixative (such as 10 % formalin) and ensure that the tissue is completely fixed to avoid tissue atrophy or deformation. (3) Tissue preservation : Ensure that the sections are preserved properly and avoid antigen degradation or denaturation due to improper preservation.
4. The antibody may cause staining failure due to insufficient affinity, cross-reaction and other reasons.

Solution : (1) Select the appropriate antibody : select the antibody that has been verified to be effective. Commercial antibodies with sufficient verification can be referred to or purchased. (2) Using a higher quality secondary antibody : The quality of the secondary antibody is very important for signal enhancement and background staining control. Use secondary antibodies with high affinity and low background. (3) Optimization of incubation conditions : Ensure that the temperature, time and buffer conditions of antibody incubation meet the requirements of antibody instructions.

reference

[1] Hussaini HM, Seo B, Rich AM. Immunohistochemistry and Immunofluorescence. Methods Mol Biol. 2023;2588:439-450. doi:10.1007/978-1-0716-2780-8_26

[2] Ramos-Vara JA. Technical aspects of immunohistochemistry. Vet Pathol. 2005;42(4):405-426. doi:10.1354/vp.42-4-405

[3] Tan WCC, Nerurkar SN, Cai HY, et al. Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond). 2020;40(4):135-153. doi:10.1002/cac2.12023

[4] Moreno V, Smith EA, Piña-Oviedo S. Fluorescent Immunohistochemistry. Methods Mol Biol. 2022;2422:131-146. doi:10.1007/978-1-0716-1948-3_9

[5] Ortiz Hidalgo C. Immunohistochemistry in Historical Perspective: Knowing the Past to Understand the Present. Methods Mol Biol. 2022;2422:17-31. doi:10.1007/978-1-0716-1948-3_2