Hello,
I am attempting to image the three compartments of the nucleolus. I have chosen RNA Pol1 as a marker for the fibrillar centre, fibrillarin as a marker for the dense fibrillar centre and B23 as a marker for the granular centre.
I have not worked with fluorescent antibodies before so I have some questions:
1) will these antibodies work with live cells or will they only work with dead cells?
2) Do I just need to purchase a antibody to the marker I am trying to locate, in this case I am using 3 markers, so 3 in total? I understand they will have to be in different regions of the color spectrum to be able to clearly distinguish them
3) Does anyone know of any other markers that can be used (relatively easily) to distinguish the the three compartments of the nucleolus?
Thank you
Hi Mohammed
I think that you want to do immunohistochemistry. There are many different protocols for this technique, but they all include:
1) fixation: this will kill your cells and stop everything inside them from moving
2) wash the fixative off your cells
3) put your primary antibody on your cells
4) wash the primary antibody off your cells
5) put your secondary antibody on your cells
6) wash the secondary antibody off your cells
Different antibodies require different fixation methods etc, so the protocol that you should use depends on the antibodies that you use. Go online and find the page that tells you all about the antibody on the website of the company that sells the antibody, and that should have references to papers that have used that particular antibody. Then you can look in the methods section of that paper to find out the best protocol.
Yes, you need a different primary antibody for every protein that you want to look at. If you want to look at them all at the same time, you will need a different secondary antibody for every primary antibody that you use. For this, you need to pay attention to the animal that the antibodies come from.
If you have any other questions, just ask.
1) Given their location deep inside the nucleus, you'll have to fix and permeabilize the cells prior to application of the antibody. (Common methods include Methanol fixation (will also permeabilize) and paraformaldehyde fixation (will require a secondary permeablilization step with something like Triton X 100). I've only seen instances of live-cell labeling with antibodies with extracellular targets.
2) If you want to multiplex all the targets simultaneously, you'll need to make sure that each of your primary antibodies are derived form different species. Has anyone ever done this sort of experiment before? Their literature might provide good guidance as to antibody selection. After you've chosen the different primaries, THEN you can choose three different colors for your secondary antibodies (I would recommend red, green, and far red). You'll need to preblock your fixed and/or permeabilized cells with serum derived from the species your secondaries were generated in. (e.g. primary antibodies from rabbit, mouse, and donkey reacted with fluorescent goat secondaries would require a pre-block with goat serum)
3) http://www.ncbi.nlm.nih.gov/pubmed/17205309
http://www.abcam.com/fibrillarin-antibody-nucleolar-marker-ab5821.html
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080237
Hope this helps!
Thank you very much Rachel and Joshua for you informative answers. They really helped.
I have read the papers you mentioned, there are over thousands of proteins localizing to the nucleolus which is a dynamic organelle, so choosing an antibody against a protein which localizes to each of the three compartments is proving quite tricky!!
So for example, for my primary antibodies, say I get antibodies against RNA Pol1 from a mouse, Fibrillarin antibodies from a rabbit and B23 antibodies from a goat, will the secondary antibodies against each of the primary ones mentioned above have to be from different species... such as secondary ab for RNA pol1 from a horse, Secondary ab for Fibrillain from a goat and secondary ab for B23 from a mouse?
If this is the case I will have to preblock my fixed cells with three different kinds of serum as my secondaries all come from three different species too? And it is the secondary ab which contain the fluorescent moiety?
I followed your link Joshua to the abcam page, the anti fibrillarin ab looks fine, but I could not find a secondary anti-fibrillarin ab, I may be confusing myself as to how this works
Thank you again for your input!!
No, your secondaries are all from the same species. E.g. in your example you would use donkey anti-mouse, donkey anti-goat, and donkey anti-rabbit and pre block with donkey serum. (secondary options from Thermo, though other vendors exist): https://www.thermofisher.com/us/en/home/life-science/antibodies/secondary-antibodies/fluorescent-secondary-antibodies.html)
You don't need a secondary anti-fibrillarin, you need a secondary that is anti- the species that generated the primary fibrilarrin antibody.
More about immunocytochemistry: https://en.wikipedia.org/wiki/Immunocytochemistry
More potentially useful information: https://en.wikipedia.org/wiki/Immunolabeling
Alternatively, if you can find your antibodies of choice that are already conjugated with fluorochrome, you don't need the 2nd antibodies. And yes if you do multiplex, you have to select the fluorochromes that their emission spectra do not overlap to each other (this depends on the filters on your microscopes too).
For staining proteins in the nucleus I would avoid big size fluorochromes such as PE (emission 575nm). Instead, you could use the synthetic one like Alexa 555. The panel of the conjugated fluorochromes could be like: Alexa 488, Alexa 555, and Alexa 647. Good luck!.
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