Is it possible to stain cells with both surface and intracellular antibodies in one step after fixation and permeabilization ? I think it makes the staining procedure shorter and may improve cell number for flow cytometer?
It depends on the antibodies that you want to use for the surface marker staining. The antibodies must be able to recognize PFA treated epitopes. You can inquire this information at the technical support of the company where you have purchased the antibodies. We are usually working with antibodies against surface epitopes of lymphocytes and APCs. Most of these antibodies do not work on the PFA treated cells.
It depends on the antibodies that you want to use for the surface marker staining. The antibodies must be able to recognize PFA treated epitopes. You can inquire this information at the technical support of the company where you have purchased the antibodies. We are usually working with antibodies against surface epitopes of lymphocytes and APCs. Most of these antibodies do not work on the PFA treated cells.
As far as I know, this is highly not recommended. While the intracellular cell staining may not be impaired, the cell surface staining may be strongly affected for at leat two reasons :
- The (difficult to predict) alteration of cell surface antigens by the fixation/permeabilization process. This is the point raised by Marcus Peters.
- The (difficult to predict) fact that antibodies used to stain cell surface antigens will actually bind accessible intracellular antigens (specifically or not).
This is why all FACS protocols recommend to perform the cell surface staining before fixation and permeabilization.
HI Alaleh Rezalotfi,
It depends on epitopes and antibodies you are using.
You have to check for the technical details of the antibody, some antibody are stable during fixation and permeabilization and some are not. Therefore you have to use stable antibody for staining after Fixation.
Not all but some of the antibody binding region (epitope) on cell surface are altered with paraformaldehyde Fixation and thus it will hinder with surface staining after fixation. you need to check whether in your staining there is alteration of antibody binding region. This you can do by comparing the two staining protocol ( 1. surface staining and then fixation, 2. Fixation and then intracellular staining). If you do not get any differences in staining patterns of the protocol, I suggest that you can use both surface and intracellular staining together.
Hope this will help you,
Gajendra
i used to stain both surface and intracellular on the same day but not in one step .
i usually do cell surface staining first then do fixation , permeabilization and ICS .
Yes you can. But as been suggested it depends on the markers that are being stained for as some epitopes are destroyed during fixation.
Agree with Marcus and Julien here. Few epitopes will retain full reactivity after any fixation, plus you will run into other issues like staining intracellularly and extracellularly at the same time instead of either one. I would strongly reccommend against doing it in one step unless it has been optimized before by someone else and you have performed a test run.
We have tested markers for staining before and after in a PFA/saponin based intracellular staining protocol and a lot of surface markers you can stain after the permeabilization step. You just need to test it for your antibodies because it's clone and fluorochrome dependent. In our hands it works for T-cell surface markers and most of the time not foor NK-cel and monocyte markers.
This a good method to employ especially if you are using a strong stimulus like PMA/Ionomycin as markers such as CD4 are readily downregulated, and it can be difficult to distinguish CD4+ T cells if utilising cell surface staining in this instance. We have done staining for CD4 and CD8 post fixing and permeabilisation in these cases and it is easier to distinguish these cells.
Thank you all from your informative comments and thank you Emily and Anne for sharing your different experiences.
I got my answer, thank you all again : )
All the best,
Alaleh
- Badges
- Science method