We have a mouse model for Alzheimer's disease which is expressing human amyloid beta in plaques. We want to detect those using a mouse anti-amyloid beta antibody, using a fluorescently labeled secondary antibody, but this gives us quite a lot of background. Especially in the blood vessels, where the secondary antibody is binding endogenous IgG molecules. I have thought of two possible strategies to overcome this, and I would like to ask you if you could help me choose the best strategy.
What I've tried so far:
Mouse anti-amyloid beta as primary antibody, donkey anti-mouse Alexa Fluor Plus 647 as secondary antibody.
What I could try:
1: the anti-amyloid beta antibody is a specific subtype: IgG2b, kappa. I can try a specific anti-mouse IgG2b secondary antibody, like this one: Goat anti-Mouse IgG2b Cross-Adsorbed, Alexa Fluor 647 (A-21242) (thermofisher.com). This should reduce the amount of background staining.
2: a mouse-on-mouse kit that blocks endogenous IgG molecules before incubation with the antibodies, like this one: Mouse on Mouse (M.O.M.) Detection Kit, Basic (vectorlabs.com), in combination with Alexa Fluor 647-conjugated streptavidin.
The second option should, in theory, give better results. But it is more expensive because I have to buy two things.
we use Rodent Decloaker, 10X - Biocare Medical for antigen retrieval, PBS-Tween 0.2% for washing, horse serum to block, and Vector TrueVIEW Autofluorescence Quenching Kit with very good results
Hey Bram, both may work. I used the MOM kit in the past, and indeed I got better contrast, but the improvement was under my expectation. The subclass specific way may also work, but the mouse may have also that subclass of endogenous immunoglobulins... Before you put too much money in this project, you may try to increase the concentration of the blocking normal serum to 10% and reduce the concentration of the primary if your signal intensity allows that. Also, look at the median eminence/arcuate nucleus area and check if you have stronger background there. You may test this also in a secondary only setup. If you get the background singal there, it is indeed the endogenous immunoglobulins, as the blood brain barrier is not there, and the MOM kit may be helpful. If not, you may have a different problem that is not necessarily related to the mouse on mouse problem. In one case after such an experience I asked a collaborator to give some Rag1 KO mouse brain sections, and it turned out that the serum cross reacted with something, becasue this mouse does not have any immunoglobulins, and the signal was still there... I sometimes get a better contrast with the citrate reteival. You will not have necessily less background, but if the signal gets stronger, you may get an acceptable contrast. Good luck! Best regards, Balazs
Last month I tried the M.O.M. kit but the results were not 100% indeed. False-positive signal was diminished, but still visible. I also tried a DIY version by incubating the tissue overnight with anti-mouse Alexa Fluor Plus 647, then the next day with anti-mouse Alexa Fluor Plus 488 (no primary antibodies). This gave comparable results to the M.O.M. kit, still some 488 signal present.
I also tried 5% BSA and 5% NDS (both separate and together), but this didn't do anything.
We're thinking on how to continue.
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