I'm not so sure that you need to change your procedure so much as to first entertain the possibility that you are looking for a one-to-one-to one correspondence between what are apples-oranges-and banana's. In-vivo there is some established, if not dynamic, level of expression of proteins including CK8. Pull those cells out and put them in primary culture and they can lose (or gain) some expressions. The very methodology you use to start the primary culture can cause loss of expression. And even if you hold the primary culture long term, no matter what you add to the culture in-vitro will never exactly parallel its in-vivo environment. The MDA's are still even more different being "not normal" in their very transformed existence. I've used MDA'a a lot and although not for CK8 expression, can tell you that what they express can certainly be different from the primary cells of their derivation or from in-vivo expression. There are ways t o check if you actually have the same "
amounts" of CK8 between MDA-231's and primary cells from a more molecular sense. It is harder than tweaking a protocol but that is where I would search first. "Very weak and obscure" CK8 in primary cells compared to primary culture cells may be true biological fact. For many things I looked for in MDA's, that was the case.
-------------- Original message --------------
From: "Sofia Havaki"
> Dear Histonetters,
> I applied immunofluorescence on primary cultures of breast cancer cells and on
> cells from the established cell line MDA-MB-231 for the localization of
> cytokeratin 8, using the primary monoclonal antibody human anti-cytokeratin 8,
> purchased from Sigma (code No: C5301). My problem is that the immunofluorescence
> protocol that I followed, gave perfect results in the breast cancer cells of the
> culture cell line, while in the cells of the primary culture it didn't work (the
> immunofluorescence was very weak and obscure).
> The cells are permeabilized with Triton X-100 0.5 % diluted in PBS for 5min.
> Could the cells of the primary culture need different permeabilization time
> compared with those of the cell line?
> Could someone help??
> Thank you in advance
> Sophia Havaki
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