In order to do anything research-based, a microscope is (usually) always a necessity.
You may be thinking that a simple compact light-microscope would do just fine; however, it's not nearly enough for any in-depth research
Thus, we turn to microscopes like fluorescent microscopes. These microscopes are magic! They allow researchers to focus on parts of their specimen/cell by staining those specific parts and nothing else.
We'll be covering exactly how this procedure is done.
What are antibodies?
There are several ways to stain specimens; however, we'll be focusing strictly on antibody staining for now. Staining with antibodies is called immunofluorescent staining!
Antibodies are proteins that look for other proteins called antigens. These antibodies SPECIFICALLY bind to this one and only type of antigen. These antibodies are crucial for the process of immunofluorescent staining.
The Procedure
Scientists first need to choose what they want to stain! So, they choose the certain antibody that would attach to the protein.
Scientists want to see what's inside the cell at a certain point in time, kind of like taking a snapshot of the cell. Thus, scientists have to first freeze the cell still! They add a special solution in the cell that essentially fixes the cell interiors in place. This stops any cellular processes or degradation that would usually happen. This also provides a lot of interesting data to the scientists. For example, scientists can stop a cell while it is undergoing mitosis to examine how the cell is actually functioning!
Now that we have fixed the cells and frozen them, the next logical step would be to add the antibodies right? Wrong. Cells are very particular and selective about what gets through their membrane, and antibodies are fairly bulky proteins that probably couldn't diffuse through the membrane.
To combat this problem, the only solution is to physically destroy the membrane by poking holes in it. This is called permeabilizing. Scientists use specific solutions in order to permeabilize the cell.
However, there's one more step before adding our antibodies! I said before that antibodies bind to specific proteins! In spite of that, antibodies can still form weak interactions with other proteins that are non-specific! These are non-specific interactions and this causes parts of the cell that the scientists don't want to see to be illuminated!
To solve this, scientists add a solution called blocking buffer, which are proteins that will bind non-specifically to anything. Thus, when you add the actual antibody, it won't bind to random proteins since the blocking buffer is already on the random proteins, hence the name "blocking" buffer!
Now comes the part where we put in the antibodies! However, this is just the first kind of antibody we put in. These antibodies are called primary antibodies. We'll implicate this later.
As we put in the primary antibodies, they will bind to their specific targets! However, if we look under the microscope, we wouldn't be able to see anything! This is because we need secondary antibodies! Secondary antibodies are the proteins that actually contain the fluorophores. These secondary antibodies are attracted to the primary antibodies! Thus, when we put in the secondary antibodies, they will find the primary antibodies and stick to them.
In the end, with the primary and secondary antibodies, we can look at the cells under the microscope to find only a few things illuminated! This proves the magic of fluorescent microscopy and why it's so influential in the scientific world!
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