Dear Everyone I am trying to set up an assay for an intracellular staining using an unlabelled rabbit polyclonal antibody, the only other option for the Ab is a polyclonal goat anti human. I am activating human Lymphocytes with PMA and Ionomycin, then permeabilizing with B& D, fix and perm, I then add the primary polyclonal Ab, followed by goat anti- rabbit secondary. The primary antibody is from Santa Cruz antibodies. I am having a lot of problems with non- specific staining between the cells and primary antibody. The secondary layer is not a problem. I have tried incubating with 20% rabbit serum with no improvement in results. The ag I am interested in staining is a small intracellular protein that is part of the lymphocyte activation pathway, and attaches to a transcellular activation marker following activation. It is part of the 'src' family of small intracellular proteins. Thanks for your help, Hilary Williams, Edinburgh University.
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