2 replies to this topic

[rajgene]

Hi All,
I am planning to do a fluorescent  Simultaneous Double Staining in lung tissue, using surface CD31(endothelial marker) and intracellular STIM1 protein. i did some background check on this and they say its ok to use a cocktail of these 2 antibodies if they are raised from different source(like rabbit and mouse). my question is how do we go about the secondary staining? do i make cocktail of secondaries and incubate the tissue  or i have to do sequential staining? any insights from people who have done Simultaneous Double Staining would be helpful.
thanks
Raj

[doxorubicin]

Hi Raj,
I have done some double staining like this before, and it is fine to mix fluorescently tagged antibodies together and to even include a DNA dye (DAPI) if you want.  Just make sure you have all the proper controls so that you know what signal is dependent on each primary antibody.  If your samples are mouse tissue, you will likely have to use a special kit for the mouse antibody to avoid secondary antibody detection of immunoglobulin present in the tissue (one such kit is called M.O.M. for mouse-on-mouse).  Also, be certain that your surface CD31 antibody is still specific once you are working with permeabilized cells/tissues....there are a lot more proteins to cross-react with in the entire cell than there are on the surface of the cell.

[rajgene]

doxorubicin, on 08 November 2011 - 03:54 PM, said:

Hi Raj,
I have done some double staining like this before, and it is fine to mix fluorescently tagged antibodies together and to even include a DNA dye (DAPI) if you want.  Just make sure you have all the proper controls so that you know what signal is dependent on each primary antibody.  If your samples are mouse tissue, you will likely have to use a special kit for the mouse antibody to avoid secondary antibody detection of immunoglobulin present in the tissue (one such kit is called M.O.M. for mouse-on-mouse).  Also, be certain that your surface CD31 antibody is still specific once you are working with permeabilized cells/tissues....there are a lot more proteins to cross-react with in the entire cell than there are on the surface of the cell.

Hey Doxorubicin,
thanks for the details. i did not think about the MOM kit until you brought it up, i will get it done. yes the mouse monoclonal CD31 antibody from BD is pretty specific and i dont think it should give a problem.
thanks again
Raj