Mouse-on-mouse (MOM) staining protocol

Last edited Wed 22 Oct 2014

Staining mouse tissue using a mouse antibody is a complicated process as it's often accompanied by high levels of background. 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.

Below we provide a few methods to try and reduce background with mouse antibody on mouse tissue staining.

Stage 1 - Methods for reducing background

Materials required

• Mouse on Mouse Polymer IHC Kit (ab269452) is a polymer-based system that provides increased sensitivity and 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.

Steps

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 time for 2 min with PBS Tween 20.

Incubate tissue sections with an unconjugated affinity purified F(ab) fragment anti-mouse IgG (H+L) for 1 hour at room temperature, or overnight at 4°C.

Proceed with antibody staining. ​

Fc receptors are present on several cell types, such as macrophages and monocytes, and can bind to the antibody and give additional background staining.

Materials required

• We recommend using F(ab) monomeric secondary antibodies to help reduce 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 as this often gives less background than using PBS-Tween 20.