The Vector® M.O.M™ Kit allows the visualization of antigens in mouse tissue sections following labeling with a mouse primary antibody. In practice, this sort of labeling can lead to high levels of non-specific background staining, as any secondary antibody used would need to distinguish between specific primary antibody and endogenous mouse immunoglobulins. Therefore, a suitable method of blocking against this background is required. The M.O.M™ Kit employs two specific agents to combat this problem – namely an IgG blocking reagent, and a mysterious solution known only as “Diluent” or “Protein concentrate”, the components of which are not stated in the specification sheet. The manufacturers also recommend the kit be used in conjunction with a biotin-streptavidin secondary-labelling kit, such as the ABC Elite Universal Kit, to optimize the signal. We have used this approach in our lab with great success.
Once the primary antibody is bound to the antigen of interest, the biotinylated “universal” secondary antibody is added (either from the M.O.M™ Kit [Biotinylated IgG reagent] or from the ABC kit [Universal secondary Ab]), forming a biotinylated antibody complex bound to the target antigen. Horseradish Peroxidase (HRP) can then be bound to this complex and visualized by adding a chromogen substrate such as diaminobenzidine tetrachloride (DAB), which results in brown staining. We commonly use DAB, and counterstain with Methyl Green (a nuclear-specific stain), which produces a nice, light green contrast in negative cells.
As with any immunohistochemical technique, sample preparation is paramount. I have used the M.O.M™ Kit on paraformaldehyde-fixed, paraffin embedded mouse and embryonic/adult zebrafish brain sections, using a mouse anti-human proliferating cell nuclear antigen (PCNA) primary antibody; I regularly observe strong, specific staining, with minimum background. The added strength of combining this kit with a biotin/streptavidin step is that given the strong affinity of avidin for biotin, the signal can be amplified many-fold over the signal generated by primary-secondary antibody interactions alone.
The specification sheet included with the kit is very thorough, and includes a simple protocol, with the additional bonus of including many troubleshooting steps and considerations for amplifying signal while reducing background. The best advice offered in the spec sheet is to use freshly made, sterile buffers and reagents wherever possible, to maximize specific signal We have also incorporated an additional “antigen-retrieval” step (using either detergent or boiling in a sodium citrate buffer), which allows better permeabilization of tissues, and is an especially effective technique if the antigen of interest is intracellular.
In summary, the Vector® M.O.M™. Kit is a very powerful method for reducing background using mouse antibodies in immunohistochemistry. The range of troubleshooting advice and protocols available with the product information is, in my opinion, first-rate, and the kit is not overly expensive. However, the fact that no avidin/biotin detection kit is included in the kit means this has to be purchased separately, adding to the cost. Furthermore, DAB is a carcinogen, which must be handled carefully. Overall though, this is a kit I use often and I have no hesitation in recommending it to others.
Graduate Student
Research Department
Peter MacCallum Cancer Centre