In my previous article, I covered different immunohistochemical staining techniques at a superficial level. In this post, I will start to explain these technologies in a bit more detail and in which situations they should be applied.
All of the following will involve additional stages when applying them, for example, serum blocking, protein blocking, avidin-biotin blocking, peroxidase blocking, and so forth, but for this article, we will concentrate solely on the principle of the detection.
The Direct Method
The direct method uses directly labeled primary antibodies. They may either be purchased already labeled or can be labeled by the researcher with the label of choice using the appropriate chemistry, such as HRP labeling.
The direct method lacks some versatility as it is not as sensitive as the so-called ‘sandwich’ methods.
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Furthermore, your primary antibody is now tagged with a label that may not be suitable for future applications. One final disadvantage is the limited range of primary antibodies that are directly labeled, and if you need to label the antibody in the lab, then this involves a time commitment.
The direct method can be used for applications such as fluorescently labeling primary antibodies in co-localization experiments where the target protein is abundant, and the primary antibody has high specificity.
A current application…watch this space!
Here’s an example of a current problem where the direct method may offer a solution. A specific application in which I am considering this approach is mouse on mouse detections (‘MOM’ i.e. a mouse monoclonal antibody applied to mouse cells or tissue). Normally, I would use a proprietary MOM kit, but I currently have a problem with an antibody! The primary antibody is giving the required staining, but is dogged by a high background. Having exhausted other options, the background staining may be eliminated by labeling the primary antibody…..watch this space. In theory, this should improve the background as there will be no ‘anti-mouse’ reagents involved in the detection, but the question is, will it have the required sensitivity?
The Indirect Method
The indirect method uses labeled secondary antibodies and has much greater flexibility than the direct method. There is a large range of fluorophore, hapten, or enzyme labeled secondary reagents available to the researcher. This means that you could detect your primary antibody in a variety of ways, depending on the endpoint that is required. One modern detection option worth knowing is polymers as secondary antibodies for immunohistochemistry.
One golden rule
The indirect method is still used extensively in flow cytometry and for western blotting as well as immunohistochemical detections. It is here that one of the golden rules of immunohistochemistry emerges; remember it well and you will never regret it. The rule is to block using normal serum from the same species as the secondary antibody.
For example, if you were using a rabbit primary antibody and a goat anti rabbit HRP secondary, you would incorporate a serum blocking step using normal goat serum before application of the primary antibody. The concentration of the blocking serum varies (between 2.5% and 20%) but the idea is to block any sites on your cell or tissue which, in this case, goat IgG would bind to. By blocking these sites with normal IgG, your labelled secondary will be unable to bind to them and will therefore prevent false positive staining.
If the direct method had the sensitivity of methods such as streptavidin or polymer detections it would be used much more extensively, but it is relatively insensitive in comparison to these techniques.
Enzymatic methods
In my opinion gaining an understanding of different detection systems and how they can be applied can give your research an advantage over your competition. By understanding the principles you can apply or adapt detection systems to meet your immediate aims or the reagents that are available in your lab.
Although you may never use a PAP method in your research, you will probably use a sandwich method with modern polymer detections. Often, these are two-step procedures, e.g., primary followed by polymer; their flexibility and versatility can be improved by using the appropriate linking antibody (assuming you know which species the proprietary polymers are raised in).
The Peroxidase anti-Peroxidase method (PAP)
The PAP method was the first sandwich method that I used and involves three main stages- application of primary antibody, secondary antibody and PAP complex. Sandwich methods are still widely used today, although the PAP method has fallen out of favor.
The PAP is an antibody-HRP complex, which increases the amount of enzyme (HRP) that can be used to detect a primary antibody. This gives it greater sensitivity than the direct and indirect methods and is used extensively in diagnostic immunohistochemistry. However, it’s a slightly inflexible method as only HRP substrates such as DAB (Brown) or AEC (Red) can be used.
The Alkaline phosphatase anti Alkaline Phosphatase method (APPAP)
APPAP is a companion to the PAP method which allows the use of different alkaline phosphatase substrates and therefore different coloured end points. In both the PAP and the APAAP the secondary is a link between the primary and the enzyme complex. For example if the primary was rabbit, the PAP complex would also be raised from rabbit, the linker would then be anti-rabbit (e.g. goat anti rabbit).
In their day the PAP and APAAP detections were very sensitive.
To push the sensitivity of the detection further, researchers looked at different ways to get more enzyme in the detection system to increase the intensity of the coloured substrate reaction. This included repeated application of the detection system by re-applying the linker and PAP (or APAAP) complex several times, and although this could improve sensitivity, background levels also tended to increase. Another modification was to use a labelled secondary and then the PAP or APAAP complex, again with the sole aim of getting more enzyme into the detection system.
Understand the ingredients to make your sandwiches more tasty!
Sensitivity does not only come from the antibody reagents. The choice of fluorochrome and substrate can also contribute to sensitivity, as does fixation, tissue preparation, and antigen retrieval. It’s a bit like cooking- by understanding your ingredients and recipes, you can adapt and modify to produce different dishes or compensate when an ingredient is missing.
The classic cheese sandwich recipe (bread/cheese/bread) and the classic immuno sandwich (rabbit/goat/rabbit or primary/link/tertiary) are pretty similar, but if you understand the principles and can use other ingredients or reagents, your options become unlimited.
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