Fluorescence Microscopy & Antibody Staining: A Step-by-Step Guide

[RPI_Quantum]

Hi, I have a question concerning fluorescence microscopy and the use of antibodies to stain cells.

I am a little confused about the acutal procedure. That is, I don't know where the antigen and the antibodies come from. Are they introduced by the experimenter, or are they naturally occurring in the cell? I know that the secondary antibody must be introduced by a person since it has the fluorescent tags on it, but I am confused about how it binds to another antibody.

If someone could just lay out the steps in the staining process in great detail, that would be a big help. I hope I presented a somewhat coherent question. I am materials scientist who is trying to become more familiar with the bio world, so perhaps my understanding seems very deficient. In any case, assistance would be appreciated!

Thanks in advance.

 

[quasi426]

Hi, I have a question concerning fluorescence microscopy and the use of antibodies to stain cells.
I am a little confused about the acutal procedure. That is, I don't know where the antigen and the antibodies come from. Are they introduced by the experimenter, or are they naturally occurring in the cell? I know that the secondary antibody must be introduced by a person since it has the fluorescent tags on it, but I am confused about how it binds to another antibody.
If someone could just lay out the steps in the staining process in great detail, that would be a big help. I hope I presented a somewhat coherent question. I am materials scientist who is trying to become more familiar with the bio world, so perhaps my understanding seems very deficient. In any case, assistance would be appreciated!
Thanks in advance.

I may state somethings that you already know, but I just want to be complete. So the antigen is a general name to the thing that the antibody binds. The binding is usually with high affinity and specificity otherwise you may have to get another antibody.

The staining process involves a primary antibody, that binds to the antigen or the molecule you are attempting to visualize. The secondary antibody binds to the Fc region of the antibody. This is basically a region that all antibodies have, a constant region. The secondary antibody is constructed to have a fluorescent molecule bonded to it, which will be used for visualization.

Why have a secondary antibody? This is because constructing a fusion protein that contains both the antigen binding site and a fluorescent molecule tagged on it is a very time consuming process. (on the order of months.) So in order to avoid this time consuming step researchers have created the secondary antibody which binds to a constant region of the primary antibody. In this scenario you can say that the primary antibody is the antigen to the secondary antibody. Hope this helps.

 

[Monique]

To answer your question of where the antibodies come from: those are raised in different organisms such as mouse, rabbit, goat, sheep or donkey. It is done by injecting the animal with an epitope of the protein of study, basically immunizing them. The animal then starts to produce antibodies that can be isolated for ascites or serum.

The actual staining procedure is the following, let's say that the primary antibody was raised in mouse and the secondary in horse, and that the protein you want to stain (the antigen) is tubulin.

- Permeabilize and fix your sample so that the protein (tubulin) is accessible to your reagents and that the sample is stable.
- Incubate the sample in the serum of the animal in which the secondary antibody was raised: horse serum (to block non-specific binding of immunoglobulin).
- Incubate the sample with the primary antibody specific to the antigen: mouse anti-tubulin.
- Incubate the sample with labeled secondary antibody specific to the primary antibody: horse anti-mouse antibody.

Then you can visualize the signal according to the kind of labeling that was used for the secondary antibody.

The reason that the staining is done in two steps is that in the way you get a very specific signal and the signal is amplified in the second step. I hope that answers your questions.

 

[RPI_Quantum]

Thank you for the responses. Both were extremely helpful. I guess all I am wondering now is how do we predict the affinity of an antibody for a given antigen? I am assuming that this is purely a chemistry question, but I'd really like to know.

Again thanks, it is nice to get help in something so new to me.

 

[Monique]

There is no way to predict the affinity. There are some handles, such that the N- and the C-terminus of a protein are most likely to be exposed to the outside and are thus available for interaction, so are the more hydrophilic parts of the protein (hydrophobic parts are buried inside). So when you want to produce antibodies, you tell bacteria to start producing the part of the protein that you think is most likely to be valuable to you. You then purify that protein (for instance with a histamine tag over a nickle column) and inject it into an animal to raise antibodies.

The serum that you collect from the animals will have a whole range of antibodies with different affinities. You can then further purify those so that you only retain the ones with the most affinity, with a series of binding and elutions for instance.

 

[Moonbear]

The reason that the staining is done in two steps is that in the way you get a very specific signal and the signal is amplified in the second step. I hope that answers your questions.

Especially with fluorescence staining, this amplification is important in order to be able to detect the signal. If you had a fluorescent tag directly bound to the primary antibody, unless you were dealing with something in very high abundance in a cell culture, you're not going to have enough molecules bound to detect it.

There are additional methods available to further amplify the signal if the indirect method (the one Monique described) is not sufficient. These involve using a secondary antibody with a biotin tag instead of a fluorescent tag, an avidin-biotin complex to bind to the biotin tag of the secondary antibody, and biotinylated tyramine that binds to the biotin on the avidin-biotin complex. Then, to detect all that, you use a fluorophore conjugated to streptavidin, which binds to the biotins exposed on the biotinylated tyramine. These many layers really amplify the signal quite robustly.

 

[RPI_Quantum]

Thanks everyone for help understanding this! Monique, your explanation of antibodies and effinity was particularly clear. Thanks Again!