I never worked with immunofluorescence (IF) before, and I need to do IF in blood cells to detect a specific DNA-RNA hybrid in nucleus. I tried to look for a protocol in the internet and in papers, but always some step is missing (for example: "cells were fixed in 4% PFA", but for HOW LONG? 10 min? 30 min?), and most part of protocols that I found are for tissues, not for cells specifically. Someone that works daily with IF could give me a hand and share a detailed protocol?
Thanks a lot in advance!
I used to do a lot of staining, but I did it on mouse brain sections. I've attached the protocol I used, but you would need to modify it for cells.
It's a pretty simple protocol. Let me know if you have any questions/need clarification!
Leonardo,
I have attached the basic immunofluorescence protocol that my lab uses for labeling frozen tissue sections or cultures of adherent cells. It sounds like you may be trying to stain blood smears or some other sort of isolated cells, rather than tissue sections, so you might need to do some modification of the protocol to make it fit your application. As you work this out, a couple of things to consider are fixation and autofluorescence.
Fixation is a balance of preserving the tissue while still preserving the antigen for labeling. For our cell culture work, we generally find that 15 minutes fixation with 4% PFA provides good preservation of the cells without harming antigenicity. This may be a good starting point to try. We use a PBS or HBSS buffer pH 7.2-7.4 final concentration of 0.1 M to make the fixative. Some antigens are very sensitive to fixation and may require a different fixation approach or very short fixation times. You may need to try a variety of conditions to optimize your labeling. I also would suggest that you use freshly made fixative made from EM quality paraformaldehyde stock. Formalin and other similar fixatives, or old fixative solutions often contain formaldehyde polymers that can cause elevated autofluorescence). If paraformaldehyde fixation doesn't work for your antigens other options include fixation in methanol and/or acetic acid which fix cells by precipitation of components rather than cross linking like paraformaldehyde.
Something annoying that we see with respect to red blood cells is that they often show very high levels of autofluorescence in tissue sections. Our protocol calls for use of NaBH4 to help reduce autofluorescence, but this doesn't seem to be as effective for red blood cells as for other cell types. In your case, since you are interested in labeling in the nucleus, this may not be a problem as mature mammalian red blood cells don't have nuclei (non-mammalian red blood cells do).
Hope this helps. Good luck.
Dave Sherry
Usually I add half of the total volume in the well of pfa 4% for 5 min, then I remove all the medium with this pfa. After that I add pfa 4% for 10 min. This helps to keep your cells attached during fixation. Then I prepare a mix solution with : triton 0.5% diluted with pbs, the antibody (you have to test the best dilution, you can try 1:200, 1:500,1:1000 and 1:2000) and normal goat serum 10%. You add this solution in the well overnight at 4 degrees. then wash with pbs 3 times for 15 min in the shaker. Add the secundary solution: triton 0.5% diluted in pbs, the secundary antibody (1:1000) and normal goat serum 10%. wait 2h at room temperature. wash with pbs 3 times for 15 min in the shaker. add Dapi or topro for nuclei stainning. and mount the lamina. if the cells are attached in the coverslip, you have to remove the coverslip , drop the mounting solution and put the coverslip on the laminula.
Hope it helps, if you have further question, I'm glad to answer.
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