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Introduction to Immunohistochemistry 

The immunohistochemistry (IHC) application requires the use of antibodies to locate and bind proteins within tissue sections. This procedure can be carried out by chromogenic detection, using a coloured substrate or detecting with a fluorescent dye.



When compared to other assays like ELISA or Western Blot, IHC is less quantitative. Instead it provides vital information regarding protein localised expression. This technique is used in both clinical and academic settings, providing necessary information, as an additional method in understanding the impact of disease and treatment in specific tissues.



Immunohistochemistry can be divided in three categories, according to how the tissue is prepared. These categories are IHC-free floating, IHC-frozen and IHC-paraffin.

Tissue processing

Fixating the tissues is a vital step for preventing the necrosis of the tissues and preserving the antigens during storage. You should establish the most appropriate IHC type for the experiment first.



Generally, paraffin-embedded is the most commonly applied, but floating or frozen sections have certain advantages, which can make them more suitable for specific experiments. Ensuring that the samples can be fixated or frozen quickly and fully after harvesting is vital for the overall quality of the procedure.

Paraffin-embedded Immunohistochemistry

The main advantage of this type of fixation is that the tissue can be stored for several years at room temperature and handling it can cause less damage to the section when compared with the other two methods.



On the other hand, gradually dehydrating the section to ensure that the paraffin penetrates it efficiently can take a long time. It is also important to note that in the event of over-fixation, the need for antigen retrieval is increased. Lastly, the formaldehyde used in the fixation step can mask some epitopes, which makes staining more challenging. 

Frozen Tissue Immunohistochemistry 

This type of IHC tissue preparation is relatively simple, consisting of freezing the sample in liquid nitrogen, dry ice or isopentane. When observing phosphorylation or other posts-translation modifications, snap-freeing is preferred.



As with paraffin fixation, frozen tissues can be stored for several years at -190° C or for 12 months at -90°C. The main advantage of this method is that it is relatively quick to perform and that it can preserve both the antigenicity and the enzyme functions. Frozen sample IHC is quite suitable for observing post-translational prootien modifications, DNA and RNA.



The most common problem with frozen tissue is the formation of ice crystals. When the tissue is not frozen fast enough crystals can form and alter the tissue structure, making this technique less suitable for studying structural morphology.



Prepared sections are also relatively thicker when compared to the paraffin embedded tissue, which can sometimes result in lower resolution.

Floating tissue Immunohistochemistry

Floating tissue preparation doesn’t require any embedding and can be stored for a short period of time at 4°C. The main advantage this method possesses over the other two is that much thicker section can be used leading to much better visualisation of the three-dimensional tissue structure.



Note that because of the thickness (over 25nm), viewing single cells and small structures is more challenging, alongside the need for clearing methods. These additional methods are sometimes needed in order to minimise light scatter and to ensure that the thicker sections can be properly imaged.

Antigen retrieval

Methylene bridges can cover up the epitopes and decrease the antigen antibody binding, so performing an antigen retrieval after the formaldehyde fixation can uncover the antigenic sites.



There are two major methods for this procedure: heat-induced and proteolytic-induced epitope retrieval.

Heat-induced epitope retrieval

The main advantage of this method is that it is a less aggressive approach and has more definable parameters when compared to the other.  This technique utilises high temperatures and buffer solutions to reverse the formaldehyde related modifications to the antigen.

Proteolytic-induced epitope retrieval 

This type is more suitable for epitopes, which are harder to retrieve. The downside is that retrieving with enzymes can result in damaged section morphology.  The enzymes that can be used for this type of retrieval are proteinase K, pepsin and trypsin.

Blocking endogenous enzymes 

When using enzymes as a detection method, blocking the endogenous ones can improve the results. A suitable time to perform this step is after incubating the primary antibody, because H2O2 treatments can possibly damage the epitopes and interfere with the binding.



Furthermore, if a HRP primary conjugate antibody is used, endogenous enzymes should be blocked prior to adding the primary antibody.

Direct or Indirect detection?

Choosing which method for detection to use during an experiment is heavily influenced by the limitations of the two methods.



Direct detection utilises a label, that has been conjugated to the primary antibody and indirect detection uses a labelled secondary antibody. This secondary antibody has been raised against the antibody type and subtype of the primary.

Direct detection

The major advantage of direct detection to IHC is that it is substantially more flexible when used in multicolour experiments. Also, this method skips the second incubation and bypasses any potential background staining. Lastly, direct detection is the preferred method when highly expressed antigens are studied.

Indirect detection

This method of detection has the advantage of signal amplification and it can successfully be used for all antigens. The only drawback to this method is that the additional steps and controls require additional care to ensure that the experiment runs smoothly. 

Choosing a primary antibody

Choosing the right antibody for an IHC experiment is one of the most important steps. Researching the specificity, what species is the antibody raised in and how well it binds to the target protein are all important factors to consider when choosing.



Sometimes knockout validation is not available, so carefully inspecting the signal-to-noise ratio is essential. Even if the antibody successfully binds the target protein, the additional noise can be too high to conduct the experiment.



If the experiment allows for an antibody, that has previously been used in IHC, to be used again, it can yield significantly better results. The main issue with antibodies, that can bind to the target protein during western blotting and using them in IHC is that sometimes they are not able to recognise the antigen, simply because it is in its native tertiary form.



Lastly, the limitations of antibody clonality can ultimately dictate your choice. Polyclonal antibodies can have high variability from one batch to the next, but they can offer much stronger signal. On the other hand, monoclonal antibodies are less prone to cross-reaction, but are much more sensitive to changes in the conditions and can easily loose their epitope following chemical treatment.



All these factors should be carefully observed so an informed choice can be made and the best experiment outcome secured.

Chromogenic and fluorescent detection

Two methods for detection are used with immunohistochemistry- Chromogenic and Fluorescent. Chromogenic detection utilises enzyme-labelled secondary antibodies and the second method uses fluorochrome-labelled secondary antibodies. The two methods have different strengths and advantages, which should be researched before choosing the type of secondary antibodies and detection methods.

Chromogenic detection

This method uses the commonly available brightfield microscope and the use of streptavidin-HRP, and biotinylated secondary antibodies can additionally increase the signal. In recent years, the use of HRP-polymer secondary antibodies is becoming more common.



The other advantage of this method is that several precipitates are photostable, which allows for the slides to be stored for a prolonged period of time.



The downside of chromogenic detection is that it takes longer than fluorescent detection. This has exceptions, but generally, chromogenic detection has more incubation and blocking steps. Also, the enzymatic amplification makes quantification significantly harder.

Fluorescent detection

Fluorescent detection can offer superior resolution and is particularly useful for visualising several antigens at the same time with several excited fluorophores at different wavelengths. This detection method takes less time to perform than chromogenic detection.



On the other hand, obtaining high resolution images requires more expensive and sophisticated equipment, making it less common. Lastly, the fluorophores can lose their stability over time. 

Multi-colour Immunohistochemistry

Staining several markers in one tissue section can be accomplished via mIHC. Usually, chromogenic multi-colour IHC depends on the antibodies to be raised either in different species or to be of different isotopes. Following this, specialised antibodies are used alongside specific chromogens for each marker. The drawback to this method is distinguishing the different chromogens on the slide, especially when more than two are use and are overlapping.



If the experiment calls for the use of three markers, fluorescent mIHC can be implemented. This method utilises either fluorescent dye-conjugated primary antibodies, or the more commonly used dye-conjugated secondaries. The reason for using these secondary antibodies is that they can offer superior amplification and are readily available, not like their primary counterparts.



Fluorescent mIHC cannot use more than three markers, because of the pre-set filter sets and antibody requirements, but there are ways to overcome these limitations. One way to do that is by the use of spectral unmixing microscopes, which allow for better distinguishing of the different dyes.



The main advantage of multi-colour immunohistochemistry is that it allows more data to be generated from one tissue section, ultimately reducing the need for several sections to be used. Furthermore, having several markers observed simultaneously allows for better understanding of their relationships and interactions. 

Counterstains and IHC

One of the most important steps of IHC is the counterstain. It is essential to visualise where the antibody staining is in relation to the tissue structures. Usually, hematoxylin is used in chromogenic IHC.



It stains the nuclei blue, which is easily distinguished from the brown colouration of HRP-DAB. For fluorescent IHC, the DAPI blue nuclear dye is commonly used.