In article <484huj$27k at server-b.cs.interbusiness.it>,
Giorgio Spagnol <spagnol at galactica.it> wrote:
>Dear fellow researchers,
>I'm studying two antibodies, which have the peculiarity of staining the
>cytoplasm or the nucleus of neural cells. They do this both on on frozen
>tissue slides and on cultured cells.
>For such a staining to occur, I presume that the antibody should first
>bind to a membrane receptor, than be internalized. This can occur when a
>Fc receptor is present, but, to my knowledge, neural cell do not have
>such a receptor. Does anybody know examples of internalization of
>antibodies outside the immune system, or can give an explanation for the
>behaviour of these two antibodies?
>I would be very grateful for any advice.
>Thanks in advance.
>Giorgio
>>
Wait a sec. It's not really clear what techniques you're using and
what you're trying to accomplish, so it's really hard to help you
trouble-shoot.
First, my interpretation of your problem is that you are trying to
perform immunohistochemistry on both frozen tissues and fresh cultures.
Is this correct? Second, are the cells fixed in any way, and where do
you actually expect the stain to be present when you are through with
your staining procedures? Are you using a fluorescently labeled reagent
or one coupled to HRP or Alk Phos?
When you get unexpected staining of the nucleus or cytoplasm, do you
mean that sometimes you get it in the nucleus and sometimes in the
cytoplasm? Sometimes both? Do the staining patterns vary from
cell to cell WITHIN a sample or are they uniform on given slide, with
the pattern varying from slide to slide? (I'm trying to figure out
if this is just background you're seeing, which seems to me to be the
most likely possibility if the molecule you're trying to detect shouldn't
even be found in the nucleus)
It seems to me that active internalization of the antibody is unlikely,
given that you also see this in formerly frozen tissues. While another
possibility is that you are capping the molecule you are trying to detect
and that you are getting internalization of your own molecule with the
antibody, it seems difficult to imagine this happening in frozen tissue
samples.
Now, assuming that you are somehow keeping your cells alive during the
staining procedure..
I suspect that active receptor uptake is unlikely to be the problem
because while that would result in cytoplasmic staining, it's hard
to imagine how those antibodies made it into the nucleus while the cells
are alive. Regardless, most of us staining live cells tend to do it on
ice (to keep the cells alive) and at that temperature, most receptor
internalization should be inhibited.
If you're using an enzyme based detection method, you should really check on
endogenous enzymes. However, I doubt that peroxidase and an alkaline
phosphatase are in the nucleus (I could be incorrect about this). If your
cells are unlikely to be alive and you are certain that the molecules you
are studying shouldn't be in the compartments you're seeing them in (not
considering things like receptor mediated internalization), then I'd be
trying to see if this is a background problem rather than some biological
process.
Again, it would really help to know the specifics of the problems you're
having before trying to narrow down the possible problems.
Stacy
