My lab acquired an interest in examining new cell proliferation in a set of developmental studies in mice via a single i.p. injection of the thymine analog 5-bromodeoxyuridine (BrdU). We have looked into a number of protocols, some of which use rat-host monoclonal primary antibodies and others that use mouse-host primary antibodies on mouse tissue. Conceptually, I have come to understand that using a single species for primary antibody host and the tissue-to-be-examined results in a lot of nonspecific background staining that makes cell counting more challenging. However, in the interest of saving, it would be great to hear that a mouse-host primary on mouse tissue labels nicely.
If you are familiar with neurogenesis IHC protocols and could recommend specific antibody products, I would appreciate the input. Any other comments on this procedure are also welcome.
Hi Steven,
we use an antibody from BD (#347580) which works really well also on mouse brain tissue. As you don´t have many endogeneous immunoglobulins in the brain, especially when you work with slices of animals that have been perfused, it is generally less problematic to use mouse antibodies on brain tissue. However, as with all things - it depends. We generally add Fab-donkey-anti-mouse fragments (1:50, Dianova) to our first blocking solution to block putative endogeneous targets for the secondary antibody. We receive very good results with little background (so little that we have to slightly counterstain with cresyl violett) using the peroxidase technique with a biotinylated secondary donkey anti-mouse Ab from Dianova (#715-065-151). In immunofluorescence we have more problems with "unspecific" binding of secondary antibodies, here we use donkey anti-ms FITC-conj., Fab (#715-097-003; 1:50). But we also have very good experience with the rat anti-BrdU (Accurate, #OBT0030CX) and almost no background. Even if there is a recent paper showing higher efficiency of the mouse ab (I don´t remember the authors...), I would recommend the rat ab, as long as you don´t switch abs within a study. Hope, this helped.