Hi all, I was just wondering if anyone has experience with multiplexing a mouse monoclonal primary and a rat primary. I'm trying to multiplex by incubating them in the same well but was told by a colleague to research the literature and find out if anyone has multiplexed with the same mouse monoclonal ab that we're buying and a rat primary. Their concern was background staining due to the species being alike (their secondaries could bind to the wrong primaries). We're using a mouse-on-mouse blocking reagent from vector. Does anyone have any experience with this or any recommendations? Thank you!
You'll want highly cross-adsorbed secondaries. Invitrogen (Thermo, or whoever they are now) sell these. They are more expensive but they are for this exact purpose. Using mouse as an example, the highly cross-adsorbed are pre cross-adsorbed against bovine IgG, goat IgG, rabbit IgG, rat IgG, human IgG, and human serum to prevent cross reactivity.
The blocking may need to be optimised as mouse-on-mouse might not work as well as a blocking reagent for other species (but adding some BSA or FBS to the blocking mixture may help). As far as the secondaries go though, highly cross-adsorbed antibodies work quite well.
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