I am doing ICC on some cultured astrocytes with an A2B5 antibody. From my understanding, this should be expressed on the cell surface and throughout the body. I have performed the stain multiple times and the fluorescent signal is localized to the nucleus. Why would this be? The antibody is older, has been sitting at 4C for over a year so I ordered new but why would it's degradation localize the staining to the nucleus the most? Thanks!
Hi Kassondra, what I think is happening is that the stored florescent antibody has aggregated and it has just precipitated over the most charged structure in the cells i.e the nucleus..
A dead giveaway is if you see unexpected punctate fluorescence dots in the cells that didn't react before.
Interesting! So by precipitating do you mean that the secondary did not bind to the primary but localized over the nucleus? In my secondary only controls there was no signal.
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