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Mouse On Mouse

Staining mouse tissue with mouse antibodies often results in high background levels due to secondary antibody binding to endogenous mouse IgG in the tissue and Fc receptors on immune cells. To reduce this background for mouse on mouse staining, endogenous IgG and Fc receptors can be blocked by incubating the tissue with serum from the same species as the secondary antibody or with anti-mouse IgG Fab fragments before continuing with antibody staining. Other tips to decrease general background include incubating tissues with Triton X-100 to clean them and using TBS-Tween 20 as the washing buffer instead of PBS-Tween 20.

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52 views

Mouse On Mouse

Staining mouse tissue with mouse antibodies often results in high background levels due to secondary antibody binding to endogenous mouse IgG in the tissue and Fc receptors on immune cells. To reduce this background for mouse on mouse staining, endogenous IgG and Fc receptors can be blocked by incubating the tissue with serum from the same species as the secondary antibody or with anti-mouse IgG Fab fragments before continuing with antibody staining. Other tips to decrease general background include incubating tissues with Triton X-100 to clean them and using TBS-Tween 20 as the washing buffer instead of PBS-Tween 20.

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lkuohg
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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MOUSE ON MOUSE STAINING

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, also Fc receptors on B cells, plasma cells and macrophages

Abcam is unable to guarantee immunostaining with 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 the only option available and you wish to attempt it:

1. Blocking of endogenous IgG:

1. Prepare tissue sections as usual.

2. At the usual blocking step, block with 10% serum (from same species as the secondary antibody) in PBS for 30
minutes at room temperature.

3. Wash 3x2 minutes with PBS tween.

4. Incubate sections with an unconjugated AffiniPure Fab fragment Anti-Mouse IgG (H+L) for 1 hour at room
o
temperature, or overnight 4 C . Try this blocking antibody at 0.1mg/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:

1. Incubate sections with 1% Triton (in PBS) at room temperature for 30 minutes to ‘clean’ the tissue.

2. Use TBS –Tween20 as a washing buffer. This often gives less background than using PBS-Tween 20.

www.abcam.com/technical

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