Immunohistochemistry (IHC) is a technique that is commonly performed in labs, and is used to identify cells or components of cells for viewing under a microscope. Here are a few sweet examples of IHC:
This picture shows villi from the small intestine, with nuclei stained red. Epithelial membranes are blue, and green labels the rapidly proliferating epithelial layer of the villi and the crypts. I found this picture on a great website called the Cell Image Library. Check it out sometime if you want to be amazed 🙂
This was taken by Jason Snyder, who has a great blog about neurogenesis which you can find here. Granule cell nuclei are labeled red, while astrocytes and radial glia are in white. Beautiful.
Last, here is a picture taken by myself of the dentate gyrus. Green labels nuclei, while red labels dividing cells (actually, dividing cells look yellow because they are both red and green). As you can see, cell division is kinda rare in the brain.
The post today is not focused on how IHC works in general, but rather the biochemistry behind it. So there will be no discussion about the difference between monoclonal and polyclonal antibodies, or what a negative control is, or the difference between primary and secondary antibodies, etc. This post will (attempt) to describe what ACTUALLY happens when an antibody binds a ligand, how EXACTLY a fluorophore works, and what fixation REALLY links.
So first, antibodies and their antigens, the celebrity couple of science. Sometimes they are together, sometimes they break up, but we always will think of them as a pair. Scientists may not know who Katy Perry is currently with, but they DEFINITELY know which antibodies go well with certain antigens, and get very upset when things don’t work out.
An antigen is a characteristic that is unique to the target you are looking for. For example, if we want to label neurons, we would find a component of the neuron that the surrounding cells don’t have.
The antibody is the labeling protein. In other words, it tags the cell you are looking for. Antibodies are very specific and will only tag the cells containing the unique antigen.
The use of antibodies in immunohistochemistry has a very interesting history. Did you know that the reason these antibodies work so well is the same reason why you won’t get chickenpox twice in your lifetime? When you get chickenpox, your body’s immune system creates antibodies that are specifically targeted to the chicken pox virus; that is, the virus antigen. After the virus leaves, your body still has those antibodies which are ready to fight the next time chickenpox tries to attack.
Antibodies for immunohistochemistry are made very similarly. The protein that you want to detect is first injected into a rabbit. Since this is not natural (just like chickenpox), the rabbit has an immune response and creates antibodies specifically targeted to the protein. Those antibodies are then collected from the rabbit and purified. Scientists then apply those antibodies to the tissue they are studying. I find it amazing that scientists have turned an already amazing phenomenon which saves people’s lives into a research tool which can then save more lives.
The main types of interactions that hold antigens and antibodies together are van der Waal forces, electrostatic interactions, and hydrogen bonds. No covalent bonds are formed when antibodies bind antigens. Van der Waal forces grow stronger when the distance between the antibody and antigen is reduced. When the structure of the antibody fits well with the antigen, the distance between them is shortened, allowing van der Waal forces to play a part. The same is true for electrostatic bonds. In electrostatic bonds, opposite charges attract. The amino acids arginine, histidine, lysine, aspartic acid and glutamic acid are electrically charged, so any protein with these amino acids can form electrostatic interactions with another charged protein.
One last bit of trivia: Temperature is an important factor in the association of antibody and antigen. This can be seen in an equation for the acid dissociation constant, KA =e–ΔaG/RT !
So that is just a little about the biochemistry behind IHC. Below are the sources that I used to create this post. If I have made any mistakes, I apologize, as I am learning this as I go along 🙂
- Boenisch. (2001) Handbook Immunochemical Staining Methods. 3rd Ed. DAKO
- Atassi, van Oss and Absolom. (1984) Molecular Immunology: A Textbook.