Immunofluorescent Staining Protocol

Immunofluorescent staining (IF) is a technique that uses antibodies and fluorophores to mark specific proteins. Scientists can use IF to identify different cell types, track the location of proteins throughout the cell, and determine protein co-localization. This insight aid in our understanding of how cells work and how they break down when we get sick.

Around the world, researchers are hard at work trying to understand, treat, and cure diseases that threaten human health. To carry out these investigations, scientists rely on a large toolbox of laboratory techniques that allow them to understand how the cells, organs, and systems in our bodies function or become sick. One tool that researchers often turn to is immunofluorescent staining. This is basically a way for scientists to visualize different proteins or structures within cells and tissues, which allows them to classify cells, monitor cellular processes, and assess the severity of different diseases. Let’s take a closer look at how immunofluorescent staining works, and then we’ll discuss how scientists could use it in their studies.

Imagine you take a vacation, and you’re flying back to your hometown at night. As the plane starts to land, you look out the window and all you can see are the lights of the city below you. It’s too dark to see any features of the land, so you try to pick out where certain parts of the city are based on the light patterns. You might be able to identify some buildings or streets, but it’s hard to decipher the full picture of where or what everything is. Now, imagine that all of the streetlights in your town used red light bulbs instead of white ones. All of a sudden, we can clearly identify where the roads and highways are because they are marked by a different color than the other lights. Next, let’s imagine that all of the big buildings in the downtown area recently switched to energy-saving bulbs that have a blue tone to them. We can now identify where the main city center is because it has a different colored marker from both the white light and the red streetlights. Imagine we kept finding ways of marking unique features of the city. It would get easier and easier to see the more complete picture of how the city looks. We could even begin to track what is happening in the city using different light markers. If all of the cars had bright green headlights on them, you could watch them travel to different places around town, or if people at a concert all turned on their phone lights at once, maybe you could identify the stadium where the show is being held. Each new marker – or different color of light – would help you visualize the structures and even activity of a city you cannot see by eye. Additionally, if you were flying somewhere else and you didn’t know which city you were flying over, having markers that highlight unique landmarks, like the Empire State Building or the Golden Gate Bridge, could even help you determine which city you’re flying over.

In the case of the immunofluorescent staining (or IF staining technique), instead of illuminating features of a city we cannot see by eye, our goal is to highlight the features of a cell that we can’t see by eye. Instead of identifying different locations or patterns in a city, we will be marking different proteins and organelles within the cell. Each marker we pick will give us different information about the identity and activity of a cell, and we can use that information to gain insight into how the cell functions under different conditions or diseases.

So, how do we actually accomplish the goal of lighting up different parts of a cell? Cells don’t have light bulbs that we can just switch out to mark different features of them, so instead we are going to utilize specialized proteins called antibodies. Antibodies bind super specifically to one protein or protein fragment inside a cell. Scientists have access to tons of different antibodies that will help provide different types of information about a cell. In our airplane example, marking the streets and buildings allowed us to visualize the landscape of the city, while marking the taxis and people at a concert allowed us to better understand the activity happening in the city. The same is true when analyzing cells. If we choose to mark different structural components of organelles, we might be able to confirm the identity or overall function, and if we mark proteins in the cytoplasm or on the cell surface, we might get a better picture of what the cell is doing.

Choosing our markers is one important facet of the process, but now let’s think about what it might take to actually gain pieces of information from these markers. Thinking back to our airplane example, if we wanted to identify where the most popular parts of the city are or where different groups of people live, it would take lots of time and effort to monitor patterns in real time. We could try and track each taxi or quickly count the number of people in an area before they move, but it would be much easier to simply take a picture of the city landscape or take pictures at different times of the night, and then use those snapshots to answer our questions about what is going on in the city.

In the case of IF staining in cells, researchers sometimes try to analyze cells in real time. Although this approach can yield a lot of interesting data, it can also be very time consuming or resource intensive, so scientists often apply the same approach of taking a snapshot of cells at a particular moment in time. To take that snapshot, researchers do something called “fixing” cells, where they apply a special solution onto the cells that essentially pauses or freezes them in time. This fixing solution stops any cellular processes or degradation that might be happening, so it becomes much easier to apply specific markers and draw conclusions about the cell.

At this point in the IF staining process, we have chosen which markers we want to use and fixed the cells in place, so they won’t move or break down. Seems like the next step would be to actually add our antibodies, but if we took a population of cells and dumped a bunch of antibodies on them, we would immediately run into a problem. Cells are designed to be very selective about what gets through their membrane and antibodies are fairly bulky proteins that can’t just diffuse through the membrane. So, how do we address this problem? Well, we’ve already fixed our cells in place, which also maintains the cell structure, so now what we can do is poke holes in the cell membrane so that the antibodies can easily pass through and enter the cell. This is called permeabilizing, and it’s important to note that this process creates holes in all membranes, so that includes the nucleus of the cell. If we tried to permeabilize the cells before fixing them, the cell wouldn’t have adequate support, so it would just collapse.

Lastly, there is one more important step we have to go through before we add our antibodies to the cells. I said before that antibodies bind really specifically to their target protein, but sometimes antibodies can form weak interactions with other proteins in the cell that aren’t the intended target. These non-specific interactions can throw off the results of your experiment if the antibody isn’t actually marking the right thing. We want to prevent this type of non-specific interaction. So, before adding the antibodies, we add a solution called a blocking solution. This contains lots of small proteins that float into the cell and occupy potential places where the antibody could bind non-specifically. It’s important to note that the blocking solution only stops non-specific interactions, so it won’t interfere with the true binding partner for the antibody we want to add.

After blocking, it’s finally time to add the antibodies. They will flow through the holes in the membrane and find their intended target in the cytoplasm or the nucleus. Once they find and bind this target, we’ve successfully marked the parts of the cell that we want to analyze. However, if we looked at the cells under a microscope, we actually still wouldn’t be able to see anything. Antibodies don’t naturally exhibit any sort of color or indicator, so the last thing we have to do is add a second set of antibodies that bind specifically to the first set we added. These secondary antibodies contain different fluorophores – or molecules that will emit a specific color when excited with a laser. We want each of our antibodies to have a distinct color, so there is no confusion about which structures are which. Let’s take a look at an example of how scientists use IF staining in a research project.

In this example, researchers are studying neurodegenerative diseases such as Alzheimer’s Disease. Since it isn’t feasible to use actual brain cells for all of their experiments, the scientists use a model called an organoid which is a small, 3D cellular model that approximates a larger organ in the body, in this case, the brain. Before they can use this model in future experiments, researchers need to validate that they have the correct cell types in their organoid model. Neurons and astrocytes are the main cell types found in the brain, so the researchers want to confirm that both of these cell types are present in their organoid. They take two small samples of the cells from the organoid and go through the process of staining one group of cells for a neuronal marker, called Nestin, and they stain the other group of cells with an astrocyte marker called GFAP. Additionally, both groups of cells were stained with a substance called DAPI, which binds DNA and helps identify the nucleus of cells.

If we first take a look at the cells stained with Nestin, we can see the DAPI stain in blue. Each dot on the screen represents the nucleus of an individual cell so we can see where each cell is in the field of view. Now, if we overlay the image of our Nestin marker on top of the DAPI stain, we can also see lots of red shapes surrounding the nuclei. The researchers used a red antibody to mark the Nestin proteins in these cells, so anywhere you see red means that Nestin is present. Nestin is a type of filament that exists in neurons, so we would expect to see red staining around the perimeter and cytoplasm of the cells, which is exactly what we see here.

Next, we can look at the cells which were stained with DAPI and the astrocyte marker. We again see the blue nuclei of the DAPI staining, and if we do the same process of overlaying the GFAP image, we can see the GFAP proteins, which have been marked by a green antibody. GFAP is a different type of filament that is associated with astrocytes, so we again expect these proteins to be present in the cytoplasm of the cells.

The fact that the organoid tissue contains cells that express both markers of neurons and astrocytes provides some support for the conclusion that the organoid is indeed a good model of the brain. The researchers will likely perform a few other tests to confirm the validity of the model before using it in future experiments, but this first IF staining experiment gave preliminary support for the model.

This is just one example of how immunofluorescent staining can be used to help researchers answer questions about the cells in our body. By using this technique to identify different cell types, track the location of different proteins throughout the cell, or determine where several proteins co-localize, researchers can build a more complete picture of how cells work and how they break down when we get sick. These insights can then aid in the development of novel therapies or approaches to the treatment of many diseases that threaten human health.

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