Hello Roxanne,

I took it from Jackson immonuresearch web site. (https://www.jacksonimmuno.com/technical/products/faq/background).

I think it may clarify your confusion.

Cross-reactivity of labeled secondary antibodies with endogenous immunglobulins on the tissues or cells.

Select a labeled secondary antibody which has been adsorbed against the species of the experimental tissue, if possible. For example, if the tissue is human tonsil and the primary antibody is made in mouse, use a labeled anti-mouse IgG that has been adsorbed against human, for example Alexa Fluor® 488-goat anti-mouse IgG (H+L) (min X Hu, Bov, Hrs Sr Prot), code number115-545-062, so that the anti-mouse IgG will not cross-react with any endogenous human IgG in the tissue.

If a mouse tissue that contains immunoglobulins is used and the primary antibody is also made in mouse, the endogenous immunoglobulins may be blocked with a monovalent Fab fragment of anti-mouse IgG. To avoid subsequent challenge by the labeled anti-mouse IgG, the tissue may be lightly fixed after Fab antibody blocking, provided this treatment does not alter the antigen to be detected. Why use an Fab monovalent antibody? If a divalent antibody is used for blocking, the second binding site on the antibody might be open to capture the subsequent primary antibody and thus amplify the background.

Sincerely,

Virginie

If you use mouse primary antibodies the secondary antibodies will have to be anti mouse (binding to mouse antibodies). As there are naturally occuring antibodies in mouse tissue the secondary antibodies could also bind to them. With rabbit primary antibodies you would use anti rabbit secondary antibody. As there are usually no natuarlly occuring rabbit antibodies in mouse tissue, the secondary antibodes have nothing to recognize (well that is not actually true, that is why you should use serum from the same animal as secondary anibody is from for blocking).

the problem is exists only if you use an antibody from an animal on tissues from the same animal. earlier this was only almost a case for mouse due to tehnique of mouse originated monoclonals only.

Since you tell us that the primary AB is IgG1, we can assume you have a monoclonal from a mouse source.   Thus your problems come from the secondary anti-mouse antibodies which contain non-specific "background" antibodies. Solutions are buy a pre-absorbed anti-mouse from Jackson et al., as mentioned above or do it yourself. For the later, you can use mouse cells or your tissue.  Simply fix cells or tissue using the protocol used for your sample, extract with MeOH, acetone, Triton which ever, then wash extensively with PBS-BSA 4-5 times (you can do this in a eppendorf and centrifuging to go faster).  Then you simply make the final dilution of your secondary AB (without DAPI) and incubate it with the cells/tissue for 30 minutes.  Your remove the later by centrifugation and use the pre-blocked secondary as you did before.   This is where the ultra-black backgrounds in IHC in the 70's and 80's came from.  Mail me if you want a detailed protocol.

Hi Roxanne,

in a previous lab we have used a M.O.M. kit to stain mouse tissue using mouse antibodies. It's from Vector Laboratories and while it's not exactly cheap, it worked quite well for our IHC (https://www.vectorlabs.com/catalog.aspx?catID=200). Maybe it's worth looking into it.