Mouse-on-mouse (MOM) staining protocol
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Tips for eliminating background when staining mouse tissue with a mouse monoclonal antibody.
Staining of mouse tissue using mouse antibody is a complicated process as high levels of background are often observed. It is notoriously difficult to eliminate this background.
Much of the background is caused by secondary antibody binding to endogenous mouse IgG in the tissue being stained, and to Fc receptors on B cells, plasma cells and macrophages.
Abcam offers a robust kit to allow use of mouse antibody on mouse tissue. Mouse on Mouse Polymer IHC Kit (ab269452) is a polymer based system that provides increased sensitivity, detection simplicity, and saves time.
Alternatively, use Goat anti-Mouse F(ab) fragment ab6668 for mouse-on-mouse blocking, and combine it with one of our Goat-anti-Mouse secondary antibodies.
Here are a few general tips to try and reduce background with mouse antibody on mouse tissue staining.
Blocking of endogenous IgG
- Prepare tissue sections as usual.
- At the usual blocking step, block with serum (from same species as the secondary antibody) for 30 min at room temperature.
- Wash 3 X 2 min with PBS Tween 20. Incubate sections with an unconjugated affinity purified F(ab) fragment anti-mouse IgG (H+L) for 1 hr at room temperature, or overnight at 4°C. Try this blocking antibody at 0.1 mg/ml although the optimal concentration will need to be assessed by the end user.
- Proceed with antibody staining.
Blocking endogenous Fc receptors
Fc receptors are present on several cell type such as macrophage, and monocytes and can bind to the antibody and give additionla background staining. We recommend using F(ab) monomeric secondary antibodies to help reduce background.
Other tips that can be used to decrease general background:
- Incubate sections with 1% Triton (in PBS) at room temperature for 30 min to 'clean' the tissue.
- Use TBS-Tween 20 as a washing buffer, this often gives less background than using PBS-Tween 20.