I am a new member to this forum. I have two main hurdles on my way to perform immunoflurescence experiments.
Firstly, the cell line which i have to use is HEK 293 cells- which seem to dettach very easily( despite of using collagen coated coverslips) during various steps of stainings especially when detergent are used.
Secondly, I need to use Flag antibody,M2(sigma) as primary antibody to stain a Flag tagged nuclear protein in my stainings which is further adding up to trouble with background staining or non-specific binding.
If anybody has any experience of Flag immunostainings for nuclear proteins and in HEK293 cells- please give your suggestions.
I wish...to get a good feedback on this query.