Mouse Antibody on Mouse Tissue Immunolabeling

Mouse-on-mouse (M.O.M.) blocking reagent prevents the binding of the secondary anti-mouse antibody to endogenous mouse tissue immunoglobulin. Essentially, if you use a primary antibody made in the mouse, the secondary antibody will need to be anti-mouse linked to a detection tag (HRP) for the secondary to recognized the primary. When you are working with mouse tissue, there are naturally occurring endogenous mouse antibodies in the tissue, which can also lead to the binding of the secondary anti-mouse HRP and the endogenous mouse immunoglobulins, leading to signal in addition to that from the primary antibody localization. To prevent this “nonspecific” staining, a mouse-on-mouse blocker is used to mask or “block” the endogenous immunoglobulins in the mouse tissue. In some experiments, we also used an unconjugated anti-mouse IgG as an additional blocking step. Because this secondary antibody is unconjugated, it will act as a place holder and will not react with a DAB substrate that produces the stain. Only the polymer-linked peroxidase-conjugated secondary antibody will react with the substrate.

mouse on mouse enzolifesciences

Photo Credit: Enzo Life Sciences

We use the M.O.M. blocker reagent from Vector Laboratories (Burlingame, CA), as it produced good results. Other commercial products may work just as well. In addition to using this blocker, we have also experimented in diluting the secondary anti-mouse antibody (1:10).  If the M.O.M. blocker was not sufficient to prevent the non-specific background stain, diluting the anti-mouse HRP secondary antibody significantly improved the specificity of immunolocalization.