Dear all,
I have a lot of non-specific bindings for one of the staining for intercellular staining in cells derived from mice. My 1' antibody is from rabbit.
If I am doing just one color for ICCS, what would be the best way to reduce the non-specific binding? Would FC block be important if I am doing perm/fix and then do ICCS?
whether you block Fc or not, you need a proper isotype control which is from rabbit and same concentration and subtype of your Ab. For me, I am bothered to block Fc receptor before I stain my cells even though they express highly.
Hi Woojong
Even though you are stainning an intracelular antigen the antibody can be recognized by Fc receptors. If your cell type express a lot of Fc receptors (macrophages for example) you should use Fc block. There is also a second possibility which is suboptimal washing. The antibody is entering the cell and can get stuck inside without recognizing the antigen. In order to evaluate this I like to have an isotype control to guarantee my cells were properly washed. Just remembering it's very important that you also titrate your antibody.
Yes , proper isotype control its good step for you
See link
Good Luck
https://www.bio-rad-antibodies.com/direct-staining-of-intracellular-antigens-by-flow-cytometry-paraformaldehyde-method.html
You may be getting high non-specific binding of your intracellular antibody, because the antibody is getting bound inside the cell. You could try and block this by purchasing a purified antibody isotype control, that is the same isotype as your intracellular antibody (i.e. purified, same type (e.g. IgG2a), but an isotype control, so it won't bind). This will hopefully saturate any sites that are picking up your antibody, giving you better staining.
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