Today is the start of a series about scientific experiments. Oftentimes in the public media, scientific discoveries are presented without explaining how scientists got those results. Understanding how experiments work is a great way to understand science in general, and also be able to spot problems or misunderstandings spread by the lay media. Today, I will focus on the use of antibodies in labs. Antibodies are essential for any lab that studies proteins. We will talk about protein visualization through the use of immunohistochemistry, protein quantification via Western blots, and finally, we will see cell identification with flow cytometry.

Before going into the experiments, one question must be answered: why antibodies? As we saw in our series on the immune system, the antibodies are able to bing a specific antigen during an immune attack. An antigen is simply a specific spot on a pathogenic protein. So scientists thought of making antibodies to be specific to a protein of interest, and nowadays, we can get antibodies for virtually any protein in any animal that we want. Another advantage of antibodies is their specificity. Theoretically, they will only bind to the protein they recognize and nothing else. While this is technically not true, as macrophage can bind antibodies for example, any other binding can be prevented during the experiment so that they will only bind the protein of interest [source].

Immunohistochemistry: how scientist see the proteins

Immunohistochemistry (IHC) is a technique that most lab will use. I will explain it through a mock experiment. I want to know where the protein adrenaline is expressed the most in a mouse brain. To do so, I will slice the brain in thin slices, and block them. Blocking is the technique that prevents any non-wanted interaction with your antibody. Then I will put my antibody against adrenaline. Now, to visualize the protein, we need a secondary antibody that is specific to the first one. Thus we have a brain slice with our primary antibody binding adrenaline, and our secondary antibody binding the primary one. Our secondary antibody was modified so that it has a fluorescent tag on it. It’s this tag that will allow us to see the protein. And now the experiment is done, we can go to the fluorescent microscope. On there, we will be able to see the proteins (in the form of coloured dots) [source].

There are many advantages to IHC, and the main one is localization. for example, in our adrenaline experiment, I will be able to see in which specific parts of the brain there is more adrenaline, and which have less. Next, it is a very easy and relatively cheap experiment. Further, we can also quantify how much protein there is by counting the dots, although this is not the best way to quantify proteins. However, the quantification can be region-specific, which is good for some proteins. IHC can also be used in more clinical applications. For example, taking tissue samples of patients, we can use IHC to determine the cancer stage for example. However, IHC also has some limitations. First, we can only use a limited number of antibodies, so we can visualize only about 4 proteins in one tissue. Further, the tissue is dead, thus we lose the protein movements. Lastly, the experiment is very sensitive to light (due to the fluorescent tag). If exposed to light for too long, then the tag will not work anymore and the experiment will be lost [source / source].

Western Blots: how scientists count proteins

Western blots are also a staple in scientific labs. I will explain it through this experiment: I want to know if stressing a mouse will increase the production of adrenaline. To do so, I will stress one mouse, and then take its tissues. I will take the tissues of another mouse that has not been stressed as a control. I will take the tissues and break them down so that we only have the proteins. Now I will separate the proteins based on size. To do so, we will perform a gel electrophoresis. We will create a special membrane in which we will put our samples. Then we will run an electric current through the membrane. Proteins are negatively charged and like a magnet, they are attracted to positive charge. This will allow the proteins to travel down the membrane. However, our membrane makes it harder for heavy proteins to travel. Thus in the end, our membrane will have all of our proteins, with the lighter ones at the bottom of the membrane and the heavier ones at the top. Next, we have to transfer our proteins on another membrane that is easier to work with and that we can keep for a longer time. This is also done with electricity. We will then block the membrane (similar to a IHC), and add the primary antibody against adrenaline, and our secondary antibody. This time however, our secondary does not have a fluorescent tag, instead it has another protein called horseradish peroxidase (HRP). This protein, when in contact with another protein called HRP substrate, will allow us to visualize our protein of interest as a black band. To do so, the substrate will be applied on the membrane, and then a film will be placed on the membrane. The film will then be exposed in a special machine so that the bands will be visible [source / source].

The biggest advantage of Western blotting is the ability to quantify. Basically, the bigger the black band, the more proteins we have. In our adrenaline experiment, the stressed mouse will have a bigger band than the non-stressed mouse. Western blots are also extremely sensitive: they can detect a very small amount of protein, which allows us to study rarer proteins more easily. However, Western blots also come with their disadvantages. The main one is its difficulty. As you may have guessed after reading the protocol, this experiment is very hard to do, and a lot of things can go wrong. It is also a very long experiment (it usually takes me two to three days to finish a blot), and it’s quite expensive as well. Another problem is the difficulty of analyzing the results. Western blots are prone to show wrong or misleading results, either because the experiment was not performed well, or because the primary antibody was not as specific as we thought. Unfortunately, there is no way of knowing what went wrong until the very end of the experiment, making this experiment very demanding [source / source].

Flow Cytometry: how scientist can identify cells

Flow cytometry is a rarer experiment. It is mostly used in immunological labs but other disciplines can also use it. It also has a big use in hospitals. Flow cytometry has many uses, but the most interesting one is to identify cells. Let’s go through it with another experiment: I want to know if immune cells are able to produce adrenaline, and if they are, which one of them are able to do so. For this, I will take a mouse and take some of their blood. I will treat the blood so that only the cells are in the sample. Then I will block it and add my primary antibodies. Here I will add antibodies for adrenaline, CD4, CD8, and B cell receptor. Then I add my secondary antibodies with my fluorescent tag. Each primary antibody has its own secondary antibody, and each secondary antibody has a different fluorescent tag. Then I can go to a machine called a cytometer. This machine will analyze the cells one by one and shine lasers on them to identify which antibody they have. We can then see which cell expresses which antibody [source].

Analyzing a flow cytometry experiment is hard. In our mock experiment, I will separate my cells into three groups: the cells that have the CD4 antibody will be CD4 T lymphocytes, the cells that have the CD8 antibody will be CD8 T lymphocytes, and the cells with the B cell receptor antibody will be B cells. Then, within each group, I can see if they express the adrenaline antibody, and how many cells within each group express it. Finally, the cytometer can allow me to take my sample back for further analysis, but if I want to, I can take only the cells that express both CD4 and adrenaline antibody, and discard the rest. As you can see, many things can be done with flow cytometry: we can identify cells, see which proteins express a specific cell, sort our cell so that our sample only contain specific cells, etc… Flow cytometry has other advantages: with the right cytometer, you can have more than 10 different antibodies, allowing you to have very specific subset of cells. Since the cytometer analyzes the cells one at a time, it is extremely specific. However, just like any experiment, there are some flaws. The biggest one is the difficulty: the experiment is very hard to perform, and the cytometer is extremely hard to use. It is also an extremely expensive experiment. Another problem is that we lose the integrity of the tissue, thus unlike IHC, we lose some of the localization [source / source].