Mouse on Mouse (MOM) staining procedure

Tips for eliminating background when staining mouse tissue with a mouse monoclonal

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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 is unable to guarantee monoclonal mouse antibodies on mouse tissue (unless stated on the datasheet). However, there are a few tips to try and reduce this background should mouse on mouse staining be necessary:

1. Blocking of endogenous IgG

  1. Prepare tissue sections as usual.
  2. At the usual blocking step, block with serum (from same species as the secondary antibody) for 30 min at room temperature.
  3. Wash 3 X 2 min with PBS Tween 20.
  4. Incubate sections with an unconjugated AffiniPure Fab fragment Anti-Mouse IgG (H+L) for 1 hr at room temperature, or overnight 4°C . Try this blocking antibody at 0.1 mg/ml although the optimal concentration will need to be assessed by the end user.
  5. Proceed with antibody staining.

2. Blocking endogenous Fc receptors

There are kits available for this which use F(ab) monomeric secondary antibodies.

Other tips that can be used to decrease general background:

Top tip Molly
  1. Incubate sections with 1% Triton (in PBS) at room temperature for 30 min to ‘clean’ the tissue.
  2. Use TBS –Tween 20 as a washing buffer. This often gives less background than using PBS-Tween 20.