Immunohistochemistry Protocol Explained in the Most Comprehensive Way

Immunohistochemistry protocol

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Immunohistochemistry protocol should be well documented in every laboratory that performs this procedure. This practice will avert errors in the process that could eventually compromise the accuracy and reliability of the test.  Compromising the accuracy of this process would mean misleading diagnosis which must be avoided.

Therefore, a standard operating procedure (SoP) or protocol for immunohistochemistry is paramount to the success of this technique during disease diagnosis.

In this article, we shall elaborately explain the protocol for immunohistochemistry. But firstly, we need to know what immunohistochemistry is and its applications as a diagnostic laboratory technique.

What is Immunohistochemistry (IHC)?

Immunohistochemistry is an immunological technique that localizes a specific antigen at the exact place where you can find it in a tissue using [antigen-antibody] reactions.

The antigen-antibody reaction is just the normal reaction like what can be performed serologically on a slide in the laboratory. The difference here is that you are performing it on the antigen in their natural location in the sample tissue.

The technique can be performed on formalin-fixed paraffin-embedded tissues. That means that the tissues can be preserved, and the procedure performed after several days while the tissue maintains a life-like state.

Analogy of Immunohistochemistry

You have a forest of trees; your workers report that some trees have what looks like a microbial infection. The exercise of spraying the whole forest is too expensive for you now. As a first step, you will ask your workers to carefully mark with paint only the trees with defects (infection) and enumerate them.

Then approach the plant infection control experts. You will request them to spray only the trees marked with a paint and not any other. The benefit of this approach is that you will know where the problem is most concentrated in addition to saving money by targeting only specific trees.

We have used the analogy of trees because they don’t move around and so once marked, then you can be sure of targeting them easily. In the same way, the antigens are marked/tagged at their exact location in the sample tissue in the laboratory using monoclonal or polyclonal antibodies then stained using dyes.

When you process the tissue and observe it under microscope, you can tell where the antigen of interest is most concentrated (because of the stain distribution) from where it is not. Other interventions can now benefit from those findings.

Applications of immunohistochemistry

In the current times when the spread of cancer is beyond what we anticipated in the past, there is, therefore, the need for accurate identification of tumor antigens. Immunohistochemistry has come in handy in this case.

Locating the exact position of tumor antigens has had value in both diagnosis and predicting the prognosis of the growth after primary interventions. An example of this in the current practice of medicine is HER2 amplification in breast cancer.

The IHC technique has now further been employed in locating the primary site of metastatic cancers. This is very important because after metastasis it can be tricky to tell what type of cancer you are dealing with, until you know the site of origin.

There are many other applications of IHC, but the focus of this article is immunohistochemistry protocol.

Step-by-step immunohistochemistry protocol

Immunohistochemistry protocol is broadly divided into two phases. These two are sample preparation and sample labeling. Each of the two phases has several steps which we shall describe here.

1. Sample Preparation in Immunohistochemistry

Sample preparation is very important because if not well done, the tissue will lose its integrity. This would interfere with the antigenic epitopes and perhaps disfigure them. If this happens, since antigen-antibody reactions are usually very specific, it will not be possible to detect the antigens in this case.

a. Collection of the Tissue

This process begins with receiving a tissue either from the theatre or from a postmortem procedure. Once received the tissue will be fixed using formalin or formaldehyde.

The tissue needs to be fixed as soon as the sample is collected to avert possible degradation and maintain its integrity in a life-like state. The tissue needs to be perfused as well.

The tissue is perfused invitro once removed but when dealing with experimental animal tissues, they can be perfused in vivo under anesthesia as well. This is done using a peristaltic pump.

The goal is to remove blood antigens and other blood components from the tissue or organ of interest. It is after this that the tissue is collected and fixed in readiness for IHC.  

b. Fixing and Embedding of the Tissue

This step is very important. The goal here is to reduce protein solubility, maintain the integrity and ensure that the antigen of interest is not trapped in a manner that would make it inaccessible to the antibody during the staining process.

Therefore, the right fixative must be used in this step. The fixative of choice that is multipurpose and fits many situations is formalin (Formaldehyde). Formalin crosslinks the components of the tissue to help them remain in a life-like state.

The good thing is that formalin can used in tissues that will be embedded using paraffin wax before they are sectioned.

Paraffin wax is again the most common agent used for embedding the tissues because it allows for antigen-retrieval using various methods that are commercially available.

Dewaxing is part of Immunohistochemistry protocol
A scientist in gloves preparing slides for Immunohistochemistry protocol (Source: Deposit Photos)

For fixation, acetone and methanol can also be used in the place of formalin and this would still give you good results. The fixatives mentioned here are all readily available commercially.

c. Tissue Sectioning and Mounting

The frozen tissue sectioning method is the most common method of sectioning. However, despite its popularity this method has the disadvantage of poor tissue morphology, decreased resolution at high magnifications, and the need for special storage of the tissues.

Here the formalin-fixed, paraffin-embedded (FFPE) tissue sections are cut into thin sections using a suitable microtome knife.

The frozen tissues in their blocks are sectioned into sections by adjusting the microtome knives as appropriate. The sections should range from 4 to 5 μm in thickness.

The sections which are dropped into a warm water bath surface during sectioning are horizontally picked using glass slides. They are then coated on the slides using tissue adhesives.

Paraffin wax step in Immunohistochemistry protocol
Two paraffin-embedded tissues in their blocks ready for sectioning

Examples of such adhesives used in this step include poly-L-lysine and 3-aminopropyltriethoxysilane. Other tissue adhesives that have been used in this step include egg albumin, gelatin, and even Elmer’s glue.

Once mounted the tissues are dried in an oven and are ready for de-paraffinization. The drying step can sometimes be avoided if it is likely to destroy the antigens of interest. The kit manufacturers will always give caution.

d. De-paraffinization

De-paraffinization is the process of removal of paraffin wax from the tissue before antigen retrieval process.  To ensure that the antigenic epitopes are available during the IHC process then paraffin should be removed completely.

If not, based on the hydrophobic nature of paraffin wax, the dyes wouldn’t penetrate in the IHC staining. The solutions used to remove paraffin from the tissue include xylene which has traditionally been used especially when dealing with FFPE tissues.

Solutions for deparaffinization are readily available commercially and so you can get them whenever you want. Their prices vary from supplier to supplier.  

e. Antigen retrieval

Antigen retrieval is an important step in IHC protocol. During fixation, what formaldehyde does is to covalently cross-link the proteins.

The cross-linked protein epitopes would not be available for antibody binding if antigen retrieval process was to be avoided.  

There are various methods used for antigen retrieval, but we explain two of them here. One is called heat-induced epitope retrieval (HIER). The other method is enzymatic antigen retrieval.

In HIER method, the deparaffinized tissues of interest (mounted on their slide) are heated or boiled in specified buffers. This is done at different pH levels. This way, the antigens are accessible for immunolabeling.

In the enzymatic method of antigen retrieval, you will treat the tissue with proteolytic enzymes that can digest the proteins in the tissue.

Examples of such enzymes include trypsin, pepsin, and proteinase K among others. Most of the antigen retrieval commercial kits available in the market will normally include an insert of how this process should be done.

In addition to our generic explanation here, you will follow instructions on such inserts. There are cases when the antigens are not hidden and so their retrieval is quite easy.   

f. Blocking Endogenous Target Proteins

To ensure that there is no false positivity in your IHC protocol results, you need to block endogenous target proteins in the tissue of interest.

Blocking will also prevent other proteins on the background from cross-reaction which is most likely make it difficult to report your results during microscopy.

Such proteins that need to be blocked include biotin and other proteins/enzymes like NeutrAvidin (NA), strept (Avidin) (SA and avidin (AV). Such proteins are blocked using a solution of appropriate antibodies and suitable buffer solutions.

Peroxidase blocking solution is very popular in this step of IHC protocol. The universal blocking buffer like Biocare Background Sniper can also be used in blocking.

Overall, what you can expect here and what your aim should be is decreased staining of the background once the tissues are treated with these solutions.

g. Blocking non-specific sites

Since antibodies can non-specifically, though weakly bind to other sites other than binding to their specific epitopes, blocking of such sites is critical in IHC protocol.

If such non-intended binding occurs, there will be high background staining, and this is likely to mask the antigen-antibody complexes of interest from being seen.

To block the non-specific sites, you will incubate the sample of interest with a buffer that blocks them. This is to make them available for binding by the primary and secondary antibodies of IHC process.

The commercially available buffers that you could use in this step include specific percentage strength of normal serum, bovine serum albumin (BSA), gelatin, and non-fat dry milk among others.

These can be used alongside gentle surfactants which helps in wetting the sample. Wetting the sample is important prior to the immunolabeling procedure.

2. Sample Labeling in Immunohistochemistry

a. Immunolabeling and Immunodetection

Since the process of immunolabelling and immunodetection is the second and last phase of IHC, you need to be careful to ensure that your efforts and processes that preceded it were not efforts in futility.

This process must be done by a qualified staff who is also highly competent. There is even the need to optimize both the primary and the secondary antibodies with buffered diluents.

The optimization helps to ensure that the diluted antibody reagents diffuse through sample tissue in a uniform and complete manner.

i) Primary Antibody Application

To begin this process, you will apply the diluted primary antibody with volume specified by the manufacturer.

You will then incubate the sample tissue at room temperature for about 1 hour 20 minutes as directed by the kit manufacturer.

The incubation period serves to allow the primary antibody to bind to the epitopes of the antigen of interest within the tissue sample on the slide.

If you are not ready to complete the process, you may incubate it at 40C overnight and resume the process the following day.

To remove the unbound material in preparation for the application of secondary antibody, you will need to wash the sample in 3 changes of 0.1% TBS-Tween.

You need to do this was as directed by the kit supplier in the paper insert. Be careful to read the insert for kit-specific details.

ii) Secondary Antibody Application

Apply the secondary antibody to the sample slide as specified by the kit manufacturer.

The secondary antibody will bind to the Fc regions of the primary antibodies to help in the identification of the antigens of interest. See the center diagram below to see how this happens.

Use of antibodies in Immunohistochemistry protocol
Images of primary and secondary antibodies in Immunohistochemistry protocol (Source: Freepik images)

After application of the secondary antibody, you need to wash in 0.1% TBS-Tween before applying horseradish peroxidase or alkaline phosphatase as specified in the kit you are using. You’ll need to wait for 15 minutes for the enzyme to attach.

The most used enzymes in IHC are alkaline phosphatase (ALP) and horseradish peroxidase (HRP), conjugated to secondary antibody reagents. When appropriate substrates for these enzymes are added there will be an enzyme-substate activity.

This will lead to precipitation lines being formed at the exact location of the antigen of interest. These precipitin lines will assume the color of the chromogen that tagged the enzyme that was added.

These precipitin lines will be visible under microscope once the background is stained as appropriate.

The alternative way of visualizing this reaction is fluorescence which is mediated by a fluorophore instead of enzyme. But the rest of the protocol would remain the same because the enzymes and chromogens are used only for visualization purpose.

b. Counterstaining of the Background

The process of counterstaining the background is to ensure that the antigen-antibody complexes that are chromogen/enzyme tagged or fluorochrome-tagged are clearly visible. Therefore, this single-step counterstaining is done after the antibody staining process is done.

The counter stains that are commonly used in this step are available commercially. Some of these counterstains include eosin, hematoxylin, DAPI, nuclear fast red, Hoechst fluorescent and methyl green stain.

c. Sealing the sample for Preservation

Once you are done with the staining part, you may seal the sample using a coverslip and a mounting solution. You could seal the cover slip using a clear nail polish-like material.

The alternative could be the use of a commercial sealant. Remember that you only seal after application of a mountant which will have cured the sample to ensure that there is no enzymatic damage.

Last step of Immunohistochemistry protocol
A female scientist ready to observe IHC slides under microscope

The sealing material including the mountant are available commercially at reasonable, value-for-money prices.

d. Sample Observation and Reporting

The slides containing the prepared samples can be viewed and reported by the following methods:

Using light microscope or fluorescence microscope where you check out for the areas of fluorescence/chromogen-stained on the tissue. The more the fluorescence, the more the concentration of the antigen of interest.

Using confocal microscopy. This will provide you with fine details of the tissue’s anatomical architecture. With this method, you will get high resolution images as well.


Immunohistochemistry protocol is a lengthy yet an important process in the diagnosis of many diseases including different types of cancer among other diseases.

The IHC protocol can be organized in two phases which are sample preparation phase and sample labeling phase where each of the two phases have several important steps.

The accuracy of IHC in the diagnosis of diseases depends on how best each step of the protocol is performed. Failure to efficiently carry out each step can result in false positive or false negative results.

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