IHC - Info

Immunohistochemistry (IHC)

Introduction

Immunohistochemistry is a laboratory technique that visualizes the existence and localization of cellular components by the use of conjugated antibodies. After their visualization these target antigens can be analysed via a microscope. This technique specifically helps in analysing the visualized protein in a cell and subcellular context.

History

First starting with the discovery of antibodies and their ability to bind specific antigens, the conjugation of dyes to an antibody led to the invention of immunohistochemistry in 1941 (Coons, Creech, & Jones, 1941).

Applications

The method of detecting an antigen via immunohistochemistry is sometimes referred to as an immunohistochemistry test. The application itself is based on thin tissue sections on a glass slide that are stained by using antibodies.

Later the application evolved and tissue microarrays were developed that even used multiple of these thin tissue section on a single slide.

Immunohistochemistry is used in diagnostics, reserach as well as drug development.

Diseases Diagnosis

One specific use of immunohistochemistry is the diagnosis of diseases by detecting specific tumor markers. Physicians are e.g. thereby enabled to descriminated between benign and malignant tumors. Further, diagnostic observations include the severity of the tumor, as well as its cellular origins in the case of metastasis leading to the localization of its primary tumor.

Diagnostic IHC markers table:

Biological Research

While immunohistochemistry is often used as a technique on its own, it is also possible to combine it with further techniques.

Its widespread uses include studies of:

Drug Development

A way of utilizing immunohistochemistry for drug development is by staining disease markers and to analyse for their up- or down-regulation.

Sample Preparation

Tissue collection and perfusion

While the visualization of the existence and localization of cellular components is an important pillar to many research areas, the collected tissue must be preserved first to stop cellular proteins from breaking down and degrading.

Before fixation or preservation, the tissue is frequently perfused in vivo, in vitro, or is simply washed to remove any blood.

This step is is intended to get rid of blood-derived antigens that can obstruct the target antigen detection.

On anesthetized animals, the animal is exsanguished with a peristaltic pump to perfuse the tissue. Then all blood components are eliminated from the whole animal or even the targeted organ (s) by rinsing the vasculature with sterile saline. As a final step, the collection and fixation of the tissue takes place.

Tissue fixation

The underlying mechanic of tissue fixation is the crosslinking of proteins and the reduction of their solubility. If this step is not performed probably, one of the most common issues is that target antigens might not be accessible to the detection antibody.

Formaldehyde (formalin) has been established as the go-to fixative. It covalently crosslinks proteins within the tissue, it is semi-reversible and can be utilized for immersion fixation or perfusion.

Afterwards, formaldehyde-fixed tissues are embedded in paraffin wax to allow for sectioning via a microtome. The cut-off slices are called formalin-fixed and paraffin-embedded or FFPE tissue sections.

Tissue embedding

Formalin-fixed tissue samples

Not only to guarantee an unaltered tissue architecture during a long-term storage but also to enable an easy tissue sectioning, paraffin is used routinely to embed formalin-fixed tissue samples.

Frozen or cryosections

Embedding the tissue in cryogenic material and snap-freezing it in liquid nitrogen represents an alternative for tissue samples that are vulnerable to chemical fixation or the step of eliminating the paraffin.

Analogous to paraffin, frozen tissue samples need to be cut into thin slices via cryostat. After the sectioning, the slices are places on slides and finally dried.

However, the method of frozen sectioning brings along disadvantags that need to be considered such as:

Sectioning and mounting

FFPE tissue sectioning and mounting

A microtome is used to slice FFPE tissue samples into thin sections betwen 4 to 5 μm. Subesequently, glass-slides are treated with 3-aminopropyltriethoxysilane (APTS) or poly-L-lysine. That leads to the presence of amino groups on the surface of the glass. Thereby, tissue that is placed on the glass slides adheres to this tissue adhesive.

Finally, after this mounting step, the sections need to be dehydrated by applying heat either through a microwave or an oven.

Frozen sections sectioning and mounting

Similary to FFPE tissue samples, frozen sections are sliced by an cryostat and afterwards placed on a glass slide with an adhesive surface. Drying of the slice is performed at room temperature usually over night.

Subsequent fixation is performed by using acetone that was cooled down to -20°C, paraformaldehyde or formaldehyde/formalin at room temperature is used.

De-paraffinization

In order to prevent hiding of the specific antigens that should be detected via the IHC staining, the paraffin within the FFPE sections has to be eliminated completly. Xylene, a flammable, toxic, and volatile organic solvent has been established as a go-to reagent for FFPE slide de-paraffinization.

Epitope (antigen) retrieval

As previously noted, the process of crosslinking proteins (methylene bridges build by formaldehyde fixation) may obscure the antigens that should be detected. Epitope retrieval is the process of making the epitops available and accessible again. While de-paraffinization is strictly necessary, the step of epitope retrieval becomes necessary only occassionaly and should be determined in the beginning of the experiments.

Heat-induced epitope retrieval (HIER)

The unmasking of the target antigens can be done heat-induction while the sample is immersed within various buffers at different pH values.

Traditionally, this is performed by pressure-boiling the tissue slides for 15-20 minutes in an acidic citrate buffer.

Proteolytic epitope retrieval

Alternatively, epitopes may also be unmasked by proteolytic digestion of the tissue sections. Commonly, pepsin, trypsin, or proteinase K are enzymes used for this process.

Quenching/blocking endogenous target activity

Biotin and its binding proteins like strept(Avidin) (SA), NeutrAvidin (NA), and avidin (AV) have been established as the most common method for amplifying the signal by increasing the amount of enzyme molecules bound to the tissue.

Further, enzyme-mediated detection of target antigens by the use of horseradish peroxidase (HRP) or alkaline phosphatase (AP) activity is commonly performed for target detection.

One of obstacle of both principles are endogenous forms of these proteins that might bind, interfere or even mask their binding partners and thus may lead to false positives. Masking them to get rid of these unwanted side-effects is referred to as inactivation or quenching.

Blocking nonspecific sites

Despite the fact that antibodies have a preference for certain epitopes, they may weakly or partially attach to non-antigen proteins that imitate the target antigen's proper binding sites.

This nonspecific binding causes high background staining as it can prevent the target antigen from being detected.

This is why buffer that prevents primary or secondary antibodies from binding to non-specific sites are employed. They consist of a standard blocking buffer which is mixed with normal serum, non-fat dry milk, BSA (bovine serum albumin), gelatin, and one or multiple gentle surfactants to help in wetting.

Sample labeling

Immunodetection - Immunohistochemistry Staining

Primary and secondary antibodies are diluted in a buffer designed to aid in antibody stabilization, encourage uniform and thorough antibody diffusion into the sample, and prevent nonspecific binding.

It's crucial to rinse the sample between applications of antibodies in order to get rid of unbound antibodies and antibodies that are only loosely attached to nonspecific sites.

Direct and indirect methods are used for antibody-mediated antigen detection.

Indirect antigen detection

The majority of indirect methods use strept(avidin) and related proteins' natural affinity for biotin to detect an antibody that has been biotinylated and bound to the target antigen.

The addition of an enzyme-conjugated strept(avidin) conjugate helps detect the antigen-bound antibody and further leads to an signal amplification.

Direct antigen detection

Direct antibody-mediated antigen detection is divided into chromogenic or fluorescent detection.

Primary or secondary antibodies conjugated to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) are used in the case of chromogenic antigen detection (Chromogenic immunohistochemistry = CIH).

These enzymes cause insoluble, colored precipitates after incubation with their specific substrates, enabling the visualization of the region of their activity. Common substrate include DAB and AEC for HRP, and Fast Red and NBT/BCIP.

Primary or secondary antibodies conjugated to a fluorophore are used in the case of fluorescence-mediated antigen detection via fluorescent microscopy (Immunofluorescence = IF).

Counterstaining

Additional staining of cell structure-specific components adds a valuable layer of visual differentiation and orientation. It is added subsequently to the primary staining.

Over the years, hematoxylin, eosin, nuclear fast red, methyl green, DAPI, and Hoechst fluorescent stain have been established as preferred counterstains.

Further, proper controls should be included in the staining set up. These include tissue-specific antigens (positive and negative control) and reagent controls (buffer/antibody diluent control and isotype control).

In immunohistochemistry, a negative control is a tissue section that is certain not to have the target antigen. This allows to exclude non-specific signals and false positive results.

Sealing the stained sample

The sample should be conserved once staining is finished in order to preserve it for future use and to avoid enzymatic product solubilization or fluorophore photobleaching.

The tissue segment and stain are secured by mounting the sample on a coverslip with the proper mounting medium (mountant).

Prolonging the fluorophores fluorescence excitation is achieved by adding an antifade reagent. Finally, sealing of the coverslip can be done by using clear nail polish or a commercial sealant.

Sample visualization

Now, the sections are ready to be analyzed and compared to other section by light or fluorescence microscopy.

Immunohistochemistry and SARS-CoV-2

Common experimental issues

Besides non-specifc staining or tissue damage, complete missing staining and high background are the most common issues.

No Staining

If there is no staining, this is usually due to an issue in the antibody-antigen binding process. They might not be compatible, their concentration may not high enough or the antibody was not stored properly.

High Background

If you see a very high background the cause might be insufficient blocking, non-specific antibody binding or the antibody concentration is to high.

Immunohistochemistry Protocol Outline

1.Tissue preparation/fixation

2.Antigen retrieval

3.Blocking of endogenous enzymes

4.Blocking non-specific binding sites

5.Primary vs. Secondary Antibody usage

6.Chromogen or Fluorochrome choice

7.Counterstain & Mount

8.IHC Control

Cell Structures and their IHC Markers

Frequently asked questions

What is the difference betweeen immunohistochemistry and immunofluorescence?

Immunohistochemistry decribes the immunological staining of tissue, while immunofluorescence describes the use of fluorophor-labelled antibodies to make target antigens visble through fluorescence.