Can’t See the Mouse for the Mice?! Solutions to Mouse-on-Mouse Immunohistochemistry Detection

The issue of mouse-on-mouse background is only a cause for concern for the histotechnologist working within a research environment. Those working in a diagnostic setting will probably never experience this as they will be working with human tissue with antibodies raised in a variety of species- but one species that won’t be used is human!

Rabbit, rat, sheep, mice…

However, within the research setting, there is a reasonable chance that at some point you will work with mouse tissue and mouse antibodies and this combination can lead to mouse on mouse background. How can this issue be overcome? Before discussing the solutions, be aware that the same issue could arise if you were working with rabbit tissue and rabbit antibodies or rat tissue and rat antibodies, or sheep antibodies on sheep tissue and so on.

Can’t see the mouse for the mice!

Because of the prevalence of mouse monoclonal primary antibodies and the use of mouse tissue, there is now a range of reagents to help overcome this challenge. The problem occurs because you are trying to detect the binding of a primary antibody raised in mouse, to mouse tissue. The issue here is that the secondary antibody, an ‘anti-mouse’ reagent when applied, in some circumstances, bind to both the mouse antibody and the mouse tissue you are working with (even if your method is correctly optimized). This results in either high background or non-specific staining.

Sometimes it works, sometimes it doesn’t

My experience over a number of years is that it’s sometimes difficult to predict when this will become a problem. I have had situations where using a mouse antibody on mouse tissue has given perfectly acceptable staining and others where the staining is completely unacceptable, factors such as tissue type, fixation, and the quality of the anti-mouse secondary antibody will probably all have an influence on the results obtained.

If it is an issue for your experiment, there are now a range of solutions available to dispel the myth that you can’t use mouse antibodies on mouse tissue.

Here are the top three solutions;

1. Use a primary raised in another species if possible. For example, the use of rabbit monoclonal antibodies is increasing, and an increasing number of protein targets can be detected using these reagents.

2. Direct detections. Use a directly conjugated mouse primary antibody. This avoids the need to use an ‘anti-mouse’ secondary thus solving the problem. However, this is limited by sensitivity, range of reagents available or requirement to label your own antibodies.

3. Use of anti-mouse IgG F(ab) blocking reagent. F(ab) fragment antibodies have only one antigen binding site. They are produced by the papain digestion of whole IgG molecules and the cleaved F(ab) fragments are purified from the Fc fragment. They are available from a range of suppliers either directly labelled (with detection reagents) or unlabelled as blocking reagents. Jackson ImmunoResearch and Abcam have suitable reagents.

Procedure example

The procedure that I would recommend would be to incubate the section with unlabelled anti-mouse F(ab) raised in the same species as the secondary antibody chosen for your detection. The theory is that the unlabelled anti-mouse F(ab) reagent will bind to the mouse tissue only therefore blocking the ability of the chosen anti-mouse secondary to bind to the tissue (as it only has one binding site it can’t bind to the mouse primary). When you apply your mouse primary, it will bind to its protein target and the anti-mouse secondary will bind only to it and not the tissue. This can then be visualised in the usual ways.

I would recommend the following steps in your protocol;

• Rabbit anti-mouse F(ab)
• Mouse primary
• Rabbit anti-mouse F(ab)-HRP
• DAB

See the Vector Laboratories website– they have a mouse on mouse detection kit which utilises this principle.

This could be made more sensitive by using a biotinylated F(ab) secondary and a streptavidin detection (bearing in mind the pitfalls of strepatavidin detections in biotin rich tissues!). Some manufacturers (e.g.the  Abcam kit and Biocare) also have mouse-on-mouse polymer detection systems. These are of course proprietary reagents and I can only guess at the composition, but they’re probably similar to the above example (an anti-mouse IgG F(ab) blocking reagent raised in the same species as the anti-mouse polymer reagent).

Finally, there are solutions to overcome these issues, so, yes mouse antibodies can be used on mouse tissue!

P.S. when using these M-O-M detections we have found that in our hands we generally need to use the primary antibody at a lower (more concentrated) dilution, than we would use for a standard detection.

Happy staining!