I am conducting an immunohistochemistry experiment using a mouse primary antibody to detect a protein in mouse brain coronal slice. The protocol that i'm using is the following: no antigen retrieval, blocking with a solution of 10% NDS, 0,3% Triton X-100 in PBS. Incubation O/N at 4°C with the primary antibody [1:100] in a solution of 1% NDS, 0,2% Triton in PBS . Sections are rinsed with PBS for 10 minutes 3 times, and then incubated with Alexa Fluor 488 anti-mouse secondary antibody, diluted [1:1000]. At the microscope I detected a green signal that correspond exactly with the signal from DAPI. I suppose that the secondary antibody stained all the cells expressing mouse immunoglobulin in the tissue, thus marking all the cells. Do you have any suggestion to avoid or reduce unspecific binding? Unfortunately I can't change the primary antibody, the only one that I found for the protein of interest is produced in mouse.
Thanks in advance for your suggestions.
To reduce non-specific binding of the secondary antibody in your immunohistochemistry experiment try to:
- Use a higher concentration of the primary antibody, like 1:50 or 1:20, so it better occupies potential nonspecific binding sites.
- Try a different secondary antibody, like a biotinylated anti-mouse followed by avidin-conjugated fluorescein. The additional amplification step may help the specific signal rise above background.
- Add mouse serum or IgG from a non-immunized mouse to the blocking buffer at 1-5% concentration. This helps block open binding sites.
- Do an absorption control by pre-incubating the secondary antibody with excess unrelated mouse IgG. This occupies its anti-mouse binding sites.
- Perform a no primary antibody control. If this shows the same pattern as your experiment, it’s definitively non-specific secondary binding.
- Try tyramide signal amplification instead of a fluorescent secondary antibody for greater specificity.
- Use a mouse-on-mouse blocking kit which provides reagents to reduce mouse IgG background.
Dear Dr. Marco Salluzzo
You should ideally choose a primary antibody raised in a different species to your sample. This allows you to avoid cross-reactivity of the secondary (anti-immunoglobulin) antibody with endogenous immunoglobulins in the sample.
Since you can’t change the primary antibody, you will have to carefully consider how you could modify your protocol to reduce background staining.
Abcam offers a robust kit to allow use of mouse antibody on mouse tissue. You may use “Mouse on Mouse Polymer IHC Kit” (ab269452) which is a polymer based system that provides increased sensitivity, detection simplicity, and saves time.
You may want to refer to the link below which provides a few general tips for reducing background with mouse antibody on mouse tissue staining.
https://www.abcam.com/protocols/mouse-on-mouse-staining-protocol
Regards,
Malcolm Nobre
In my hands the M.O.M. Kit always worked quite nicely (see link attached). I have always worked with muscle but I do not see any reason why this kit should not work on brain sections.
I hope this helps :)
https://vectorlabs.com/products/mouse-on-mouse-basic-kit/
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