Immunohistochemistry is both an experimental and diagnostic technique which is frequently used in research and clinical pathology laboratories. In this blog, I will provide an introduction to immunohistochemistry, describe how it is used to diagnose neurodegenerative diseases post-mortem, and give examples of how it has been used in my own experimental research.
So what exactly is immunohistochemistry? Firstly, some biology. Our organs comprise of different tissue types, which in turn comprise of different cell types. These cells will express various proteins, which, in this context, we can call antigens. In simple terms, immunohistochemistry is the method of using antibodies to bind to specific antigens to allow us to visualise certain cells, or the proteins expressed by them. This visualisation is possible because when the primary antibody binds to the antigen in the tissue, a secondary antibody is then used to bind to the primary antibody which can be conjugated to an enzyme that produces a colour following a reaction (known as chromogenic immunohistochemistry). The colour produced depends on the chromogen used, but the one most commonly used will produce positive brown staining. If the secondary antibody is conjugated to a fluorophore, you get very cool and colourful immunofluorescent staining. These staining methods are typically done on thin tissue sections which are embedded in paraffin wax, which is removed prior to the staining. When the staining protocol is completed, a coverslip is placed on the slide, and it can then be viewed under a microscope.
There are many more steps involved in this process, but hopefully this gives a basic understanding of the theory underlying this technique. Having a good working knowledge of immunohistochemical theory is essential for any scientist working in pathology, and I’ve learnt that this can lead to various superstitions. Any basic immunohistochemical protocol will have the same core steps outlined above, but there is flexibility for some steps. For example, some protocols may require a primary antibody to be incubated at room temperature for one hour, but another may state that it needs to be overnight at 4oc (i.e., in a cold room or a fridge). This is even if in both cases the same antibody was being used on the same tissue. How can this be? Having worked in three pathology labs during my research career, I have never used the same protocols for the same antibodies when moving between labs. Usually, established labs will have tried and tested protocols for many commonly used antibodies which have gone through a process called optimisation. This is where each step of the protocol has been tweaked to maximise the positive specific staining, and minimise any background staining, which can happen when there is weak binding to non-specific proteins which may have similar bindings to the target antigen. Many steps in the protocol can be affected by things like temperature, humidity, and agitation of the tissue whilst it is incubating in the solutions. This is why there is an element of superstition in immunohistochemistry, where if you know a certain protocol works in a particular lab, you stick with using that, even if you have a working protocol that you’ve used in a previous lab.
Having a good working knowledge of immunohistochemical theory is also important because there will inevitably be a degree of troubleshooting required for any scientist doing pathology. This will primarily be part of the process of optimisation if you have a new antibody you want to use for the first time in your lab, so you have no established protocol for it. But sometimes, often for no apparent reason, your staining fails to work. You may fail to get any staining, or you may have too much background staining, and this can even be when you’ve used that protocol before, and no conditions have changed. As you cannot visualise the staining until the last step because this is when the colour reaction happens, you will not know until the end of the protocol whether or not the staining has worked as expected. So depending on what the outcome is, you will need to use your knowledge to determine at which step the process may have failed.
So I’m sure the question you’re wanting to ask me now is: why would anyone use this technique, it sounds like a big headache! This is certainly true, and I’ve lost many days and weeks to optimising protocols and troubleshooting. But immunohistochemistry is essential for both diagnostic and experimental pathology. Neurodegenerative diseases, like Alzheimer’s, can only be definitively diagnosed following neuropathological assessment post-mortem, where brain tissue samples can be stained for the pathology which is characteristic of the disease, such as the presence of amyloid plaques and tau tangles. Neuropathological assessment is therefore heavily reliant on immunohistochemistry to determine the presence or absence of particular disease pathologies.
In addition to diagnostics, immunohistochemistry is also used in experimental pathology. For example, I have used it to track the progression of disease pathology in an animal model, and to determine the characteristic pathological features of a neurodegenerative disease where consensus criteria is newly established.
Despite the challenges associated with using immunohistochemistry, and the superstitions of the scientists that use it, the applications of immunohistochemistry in both diagnostics and experimental research are clear. No other methods can be used to replace it, making it an invaluable scientific technique for understanding disease pathology.
Dr Kamar Ameen-Ali is a Lecturer in Biomedical Science at Teesside University & Affiliate Researcher at Glasgow University. In addition to teaching, Kamar is exploring how neuroinflammation following traumatic brain injury contributes to the progression of neurodegenerative diseases that lead to dementia. Having first pursued a career as an NHS Psychologist, Kamar went back to University in Durham to look at rodent behavioural tasks to completed her PhD, and then worked as a regional Programme Manager for NC3Rs.