I am using Immunostaining to evaluate a cell surface expression of one protein. when I use non-permeablization technique to just stain the cell surface localization (fixed cells with PFA , 2% 5 min), there is staining of protein of interest in the nucleus! Do you have any suggestion what is the reason? really appreciate your help
It is difficult to point to the exact cause of non-specific nuclear staining, but a few things to consider are:
Are the cells alive? If the cells are dead and the membrane integrity is lost, then your fluorescent antibodies could be bound to DNA, which is sticky. Incorporating a live/dead dye would be helpful to exclude dead cells. If the nuclear staining is only present in some of your cells, this is what I would suspect is a likely scenario.
Which fluorophore are you using? Some channels such as green tend to have a high autofluorescence. If the nuclear staining in all of your cells, it could be something related to this.
Good luck!
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