live cell immunofluorescence - is my thoughts correct (Jul/20/2005 )
I am a layman to biology. So this question may be too simple for you guys. But I need an answer.
Most immunofluorescence work I found use fixation before applying the antibody. But in my case, the protein is a membrane protein. So I am think if I can skip the fixation and apply the primary and secondary antibodies directly. Will this cause some problem?
Even simpler, is there some way to stain cell membrane while not disturbing the viability?
Is it crucial not to disrupt the viability? I routinely stain cell membrane associated antigens on cell cultures after fixing briefly in PFA with no problem.
If desired you can fix after staining but I would keep everything on ice to prevent internalization (this is a concern in my case, may not apply to your system).
There is normally no reason not to fix first unless your antibody will not react with fixed proteins.
then it is not 'living cell'. Or at least not cells at physilogical conditions.
If desired you can fix after staining but I would keep everything on ice to prevent internalization (this is a concern in my case, may not apply to your system).
There is normally no reason not to fix first unless your antibody will not react with fixed proteins.
True, but that is sort of the whole point ...
The cell doesn't need to be living to stain the membrane and the results to be meaninful. The whole point of fixation is to preserve the cell in the last state it was in at the time you want to manipulate it so you can do further manipulations (for example membrane staining) without worrying about the cell, proteins etc "changing". I recommend using a protein crosslinker such as PFA at a low percentage.
If you are insistant on doing live cell staining, it totally works, just do it on ice. But beware that if your intention is to continue culturing them after staining, you must make sure to maintain sterility at all stages including visualization. Also be aware that adding antibody may havea deleterious effect on cells depending on what type of cell it is. If your intention is not to culture them I would then recommend you fix at the end of the staining protocol to preserve the staining, otherwise it is pretty meaningless unless you keep everything cool and take pics ASAP.
What you said is correct, but I need to observe some reactions after staining. So I can not fix the cells.
Putting on ice is the way that I used to do. However, many reactions stop at this temperature. So I can not do it on ice either.
Any other suggestions? Thank you.
True, but that is sort of the whole point ...
The cell doesn't need to be living to stain the membrane and the results to be meaninful. The whole point of fixation is to preserve the cell in the last state it was in at the time you want to manipulate it so you can do further manipulations (for example membrane staining) without worrying about the cell, proteins etc "changing". I recommend using a protein crosslinker such as PFA at a low percentage.
If you are insistant on doing live cell staining, it totally works, just do it on ice. But beware that if your intention is to continue culturing them after staining, you must make sure to maintain sterility at all stages including visualization. Also be aware that adding antibody may havea deleterious effect on cells depending on what type of cell it is. If your intention is not to culture them I would then recommend you fix at the end of the staining protocol to preserve the staining, otherwise it is pretty meaningless unless you keep everything cool and take pics ASAP.
Have you even checked to see if you can label your proteins live? Some epitopes require fixation to be exposed for proper antibody binding.
Putting on ice is the way that I used to do. However, many reactions stop at this temperature. So I can not do it on ice either.
Any other suggestions? Thank you.
True, but that is sort of the whole point ...
The cell doesn't need to be living to stain the membrane and the results to be meaninful. The whole point of fixation is to preserve the cell in the last state it was in at the time you want to manipulate it so you can do further manipulations (for example membrane staining) without worrying about the cell, proteins etc "changing". I recommend using a protein crosslinker such as PFA at a low percentage.
If you are insistant on doing live cell staining, it totally works, just do it on ice. But beware that if your intention is to continue culturing them after staining, you must make sure to maintain sterility at all stages including visualization. Also be aware that adding antibody may havea deleterious effect on cells depending on what type of cell it is. If your intention is not to culture them I would then recommend you fix at the end of the staining protocol to preserve the staining, otherwise it is pretty meaningless unless you keep everything cool and take pics ASAP.
we do all our live cell with cells we have tagged via transfecton..i.e. SK-Texas Red or SK-GFP. If you are using a cell line it works great and doesn't have the effects of ab interaction with the protein you are looking at and causing downstream signaling.